System and method for sorting particles

ABSTRACT

A multi-channel system for classifying particles in a mixture of particles according to one or more characteristics including a common source of electromagnetic radiation for producing a beam of electromagnetic radiation and a beam splitter for producing multiple beams of electromagnetic radiation for directing multiple beams of electromagnetic radiation to each interrogation location associated with each flow channel of the multi-channel system.

This application is continuation of U.S. patent application Ser. No.16/155,576, filed on Oct. 9, 2018, now U.S. Pat. No. 11,104,880, whichis continuation of U.S. patent application Ser. No. 15/179,722, filed onJun. 10, 2016, now U.S. Pat. No. 10,100,278, which is continuation ofU.S. patent application Ser. No. 14/683,936, filed on Apr. 10, 2015, nowU.S. Pat. No. 9,377,939, which is a continuation of Ser. No. 14/206,832,filed on Mar. 12, 2014, now U.S. Pat. No. 9,040,304, which is acontinuation of Ser. No. 13/762,003, filed on Feb. 7, 2013, now U.S.Pat. No. 8,748,183 which is a continuation of U.S. patent applicationSer. No. 13/422,705, filed Mar. 16, 2012, now U.S. Pat. No. 8,535,938,which is a continuation of U.S. patent application Ser. No. 13/106,671,filed on May 12, 2011, now U.S. Pat. No. 8,206,987, which is acontinuation of U.S. patent application Ser. No. 12/794,921, filed onJun. 7, 2010, now U.S. Pat. No. 7,943,384, which a continuation of U.S.patent application Ser. No. 10/812,351 filed Mar. 29, 2004, now U.S.Pat. No. 7,758,811, which claims priority from U.S. Patent ApplicationNo. 60/458,607 and U.S. Patent Application No. 60/458,731, both filedMar. 28, 2003. The entire disclosure of each application is incorporatedherein by reference.

BACKGROUND OF THE INVENTION

This invention relates generally to apparatus and methods for animalsemen collection, and more particularly to apparatus and methods usingvarious techniques, including flow cytometry, to yield sperm populationsthat are enriched with sperm cells having one or more desiredcharacteristics, such as viable populations of sperm cells sortedaccording to DNA characteristics for use by the animal productionindustry to preselect the sex of animal offspring.

The fertilization of animals by artificial insemination (AI) and embryotransplant following in vitro fertilization is an established practice.In the livestock production industry, the ability to influence thereproductive outcome toward offspring having one or more desiredcharacteristics has obvious advantages. By way of example, there wouldbe an economic benefit in the dairy industry to preselect offspring infavor of the female sex to ensure the production of dairy cows. Effortshave been made toward achieving this goal by using flow cytometry tosort X and Y sperm cells, as evidenced by the disclosures in U.S. Pat.No. 6,357,307 (Buchanan, et al.), U.S. Pat. No. 5,985,216 (Rens, etal.), and U.S. Pat. No. 5,135,759 (Johnson). However, none of theseefforts has resulted in the introduction of a commercially successfulhigh-throughput system capable of producing production volumes ofrelatively pure sexed sperm cells having a motility sufficient foreffective fertilization.

Accordingly, there is a current need in the animal production industryfor a viable high-speed system for efficiently isolating sperm cellsbased on a specified DNA characteristic (or other characteristics) toproduce quantities of such cells, which can be used on a commercialscale. Also needed is a sperm handling system that preserves theviability of such isolated sperm as it is processed by the isolatingsystem and that allows for preservation of such isolated sperm untilsuch time that it is ready for use. The present invention addressesthese needs.

This invention also has application to improvements in the field of flowcytometry on a more general basis. Flow cytometry may broadly be definedas measuring characteristics of individual particles as they passgenerally single file in a fluid stream through a measuring devicewhich, typically, provides information for classifying the particlesaccording to selected characteristics. Optionally, the particles maythen be separated into populations using any number of techniques,including droplet sorting, droplet interference sorting, and fluidswitching. Another option is to selectively destroy unwanted particles,for example by photo ablation.

In an optically-based flow cytometry system, optics are used to directand focus a beam of light (e.g., visible light or UV light) on thestream containing the particles, and to collect emissions from theparticles, including scattered light and/or fluorescence emissions fromthe particles. In one common optic system, for example, a beam of light(e.g., a laser beam) is focused on the stream and emissions arecollected by a pair of collection units, one positioned forward of thelaser for collecting scattered light emissions and another positionedorthogonally to both stream and the laser for collecting fluorescenceemissions. Each collection unit includes a separate photodetector, whichincreases the cost of the system. Further, in traditional optic systemsthe photodetectors translate the collected emissions into electricalsignals, which are analyzed using analog systems to classify theparticles according to selected characteristics of the particles. Analogsystems are relatively inexpensive, but only limited information can bederived from the signals.

Others have tried to develop technology that can be used to processsperm cells to obtain populations of sperm cells that are enriched withsperm that have a desired sex chromosome. However, the existingtechnology falls short of the inventive technologies described herein.

For example, Johnson et al. (U.S. Pat. No. 5,135,759) describe theseparation of intact X and Y chromosome-bearing sperm populationsaccording to DNA content using a flow cytometer/cell sorter into X and Ychromosome-bearing sperm enriched populations. As described, the spermis combined with a DNA selective dye at a temperature of 30 to 39° C.for a period of 1 hour (39° C.) to 1.5 hours (30° C.). A flow cytometeris then used to measure the amount of fluorescent light emitted as thesperm passes through a laser beam that excites the dye. Because the Xchromosome-bearing sperm contains more DNA than the Y chromosome-bearingsperm, with most species of mammal having about 3 to 5% difference, theX chromosome-bearing sperm emits more fluorescent light than the Ychromosome-bearing sperm. In order to account for the fact that thefluorescence measurement may vary depending on the rotationalorientation of the sperm cells, two photo detectors are used. The firstdetermines whether the sperm cells are properly oriented, while thesecond takes a measurement that is used to classify the sperm as havingan X or Y chromosome. An oscillator is used to cause the streamcontaining the sperm to break into droplets downstream of the placewhere the sperm pass through the laser beam. Droplets containing singlesperm of a predetermined fluorescent intensity are given a charge andelectrostatically deflected into collection vessels. The collected,gender enriched sperm population, is then used for microinjection, invitro fertilization, or artificial insemination.

Seidel et al. (WO 02/43574) also describe separation of sperm intogender enriched populations of X and Y chromosome-bearing cells usingflow cytometry. Seidel et al. describe staining the cells at atemperature between 30° C. and 40° C.

United States Patent Application Pub. No. 2003/0157475 A1 (Schenk, Aug.21, 2003) describes a method of cryopreserving sperm cells that havebeen sorted according to X or Y chromosome content. As noted therein, itis desirable to add a cryoprotectant to sperm cells before they arecryopreserved to protect the sperm cells during the cryopreservationprocess. For example, glycerol is one cryoprotectant that is commonlyadded to bovine sperm cells prior to cryopreservation. However, in orderto obtain better protection from the cryoprotectant, it is desirable towait for the cryoprotectant to equilibrate with the sperm cells beforesubjecting the sperm cells to temperatures below 0° C. During theequilibration period, the cryoprotectant penetrates the cell membrane toprovide intra-cellular protection in addition to any extra-cellularprotection provided by the cryoprotectant. Thus, the cryopreservationmethods described in United States Patent Application Pub. No.2003/0157475 A1 specify that an extender containing glycerol is added tothe sperm cells after they have been cooled to about 5° C. Then thesperm cells and glycerol are allowed to equilibrate at 5° C. foranywhere between 1 and 18 hours before the sperm cells are subjected tolower temperatures. The disclosure recommends an equilibration period ofbetween three and six hours in order to obtain the best results.

Unfortunately, the time and expense involved in a 3 to 6 hourequilibration period will have a negative impact on profitability of acommercial sperm sorting process. Furthermore, in the context of acommercial sperm sorting process, it is believed that the health of thesperm is generally improved by reducing the time between collection ofthe sperm and cryopreservation (other factors being equal). From thisstandpoint as well, it would be desirable to have access tocryopreservation technology that does not require a long equilibrationperiod to obtain the optimal benefits of a cryoprotectant. Moreover, theknown cryopreservation technology is reported to have a detrimentalimpact on sperm motility, which is indicative of decreased spermfertility. Thus, there is a need for cryopreservation techniques thatpreserves sperm health compared to conventional techniques.

SUMMARY OF THE INVENTION

This invention is directed to an improved system (methods and apparatus)for analyzing, classifying and sorting particles based on one or moredesired characteristics; the provision of such a system which, in oneembodiment, uses flow cytometry to accurately isolate and sort cells byDNA content; the provision of such a system which, in certainembodiments, incorporates sorting protocols which enable the output ofthe system to be controlled as a function of one or more factors,including the purity of the desired sorted population of particles, therate at which the desired particle population is collected, the loss ofdesired particles not sorted into the desired population, and otherfactors; the provision of such a system which, in one embodiment,operates at high-speed to provide sex sorted sperm for commercial use bythe animal production industry; the provision of such a system which canbe used to sort cells without significant detrimental effect on thecells, including the motility of sperm cells; the provision of a systemthat can be used to preserve sorted sperm cells until they are neededwith minimal detrimental effect on the cells, including, the motility ofthe cells, the provision of such a system which, as it relates to theproduction of sexed sperm, incorporates techniques which increase thespeed and accuracy of the classification and sorting of the sperm cells;the provision of a flow cytometry system which uses epi-illuminationoptics to detect various characteristics of particles to be analyzedand, optionally, sorted; the provision of such an epi-illumination flowcytometry system which is economical to manufacture; the provision of asystem which, in one embodiment, incorporates multiple flow cytometryunits which share an integrated platform for classifying and(optionally) sorting particles, such as cells in general and sperm cellsin particular, at high rates of production; the provision of such amulti-channel system which shares common components and systems toreduce variations between the channels for more efficient operation; andthe provision of such a sorting system which, in one embodiment,incorporates protocols which enable a sample to be quickly tested todetermine the quality of the sample so that the profitability of furthersorting can be evaluated.

In addition, this invention is directed to an improved system (methodsand apparatus) for digitally processing signals representingfluorescence; the provision for such a digital system, in oneembodiment, for detecting analog to digital converted-pulses as afunction of background characteristics; the provision for such a digitalsystem, in one embodiment, for initializing discrimination parameters;the provision for such a digital system, in one embodiment, fordetecting digital information corresponding to waveform pulses; theprovision for such a digital system, in one embodiment, for digitalinformation analysis including feature extraction; the provision forsuch a digital system, in one embodiment, for classifying pulses anddefining decisions boundaries; the provision for such a digital system,in one embodiment, employing a droplet break-off sensor to controltransducer amplitude; and the provision for using such a digital system,in one embodiment, to distribute and collect cells for commercialdistribution.

Further, this invention is directed to an improved comprehensive system(apparatus and methods) for commercial processing of animal semen fromthe time a semen sample is collected from a male animal throughcryopreservation of a sperm sample containing a greater percentage of asperm having a desired chromosome characteristic than exists in thecollected semen; the provision of such a system, in one embodiment, thatallows efficient processing of commercial quantities of viable genderenriched sperm; the provision of such a system that allows, in oneembodiment, adjustment of the system to counter day-to-day andanimal-to-animal variations in the semen characteristics; the provisionof such a system that, in one embodiment, allows production of about18,000,000 gender enriched sperm per hour by a single flow cytometryunit at 85% purity; and the provision of such a system that allows, inone embodiment, complete processing of a batch of semen (e.g., theamount of semen collected from a male animal) to yield viable spermsamples having a desired gender characteristic at 85% purity with lessthan 10% loss of collected sperm having the desired gendercharacteristic in about 1 hour of processing time.

In general, this invention is directed to the apparatus and methods setforth in the claims of this application.

Other objects and features of this invention will be in part apparentand in part pointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a work flow diagram for an exemplary sperm sorting process ofthe present invention;

FIG. 2 is a schematic diagram of one embodiment of a flow cytometrydroplet sorting system of the present invention;

FIG. 3 is a side view of a portion of one embodiment of a flow cytometryapparatus of the present invention for droplet sorting showing anepi-illumination optic assembly focusing an excitation beam on an upwardmoving fluid stream generated by a nozzle system;

FIG. 4 is an end view of one embodiment of a nozzle and nozzle holder ofthe present invention;

FIG. 5 is a sectional view of the nozzle and nozzle holder of FIG. 4taken through cutting plane 5-5 on FIG. 4;

FIG. 6 is a schematic diagram of a sperm cell entrained in a fluidstream being interrogated by an elliptically shaped beam spot accordingto one embodiment of the present invention;

FIG. 7 is a schematic diagram showing the angular envelope for thedesired orientation of a sperm cell in which the light beam from theoptics system will strike a wide face of the cell generally broadside;

FIG. 8 is a cross sectional view of one embodiment of a nozzle body ofthe present invention;

FIG. 9 is a side view of the nozzle body shown in FIG. 8 showing aseries of cutting planes (A-A through H-H and J-J through K-K) throughthe nozzle body;

FIGS. 9A-9H and 9J-9K are sectional views of the nozzle body shown inFIGS. 8 and 9 along the corresponding planes (A-A through H-H and J-Jthrough K-K) of FIG. 9;

FIG. 10 is a perspective view of a cross section of one embodiment of anozzle system having an orienting baffle in the nozzle;

FIG. 11 is a cross sectional view of the nozzle system shown in FIG. 10;

FIG. 12 is an enlarged partial cross sectional view of a portion of thenozzle system shown in FIGS. 10 and 11;

FIG. 13 is an enlarged partial cross sectional view similar to the viewshown in FIG. 12, but taken from a direction that is perpendicular tothe viewing direction in FIG. 12;

FIG. 14 is a side view of one embodiment of baffle holder holding abaffle plate;

FIG. 15 is a top view of the baffle holder and baffle plate shown inFIG. 14;

FIG. 16 is a top view of one embodiment of a baffle holder rotationallyoriented in a nozzle so that the legs of the baffle plate intersect in aline that is parallel to the major axis of ellipse D in the nozzle;

FIG. 17 is a top view of one embodiment of a baffle holder rotationallyoriented in a nozzle so that the legs of the baffle plate intersect in aline that is perpendicular to the major axis of the ellipse D in thenozzle;

FIG. 18 is a side cross sectional view of one embodiment of a nozzlesystem including a baffle showing a series of cutting planes (A-Athrough E-E) through the nozzle and baffle;

FIGS. 18A-18E show the cross sectional flow areas at various points inthe nozzle system shown in FIG. 18;

FIG. 19 is a cross sectional view similar to FIG. 12 taken through anozzle having a baffle plate that is perpendicular to the longitudinalaxis of the nozzle;

FIG. 20 is a cross sectional view of the nozzle shown in FIG. 19 takenthrough the cutting plane 20-20 shown on FIG. 19;

FIG. 21 is a cross sectional view similar to the cross sectional view ofFIG. 18 showing a nozzle system having a sample introduction conduit atan offset location;

FIG. 22 is a perspective view of one embodiment of a nozzle systemmounted on a nozzle mount of the present invention;

FIG. 23 is schematic diagram of a plurality of aligned sperm cells beingrotationally oriented as they pass through an orifice member of thepresent invention toward the interrogation location;

FIG. 24 is a schematic diagram showing the droplet break-off locationdownstream from the nozzle according to one embodiment of the presentinvention;

FIG. 25 is a schematic diagram of one embodiment of a break-off sensorsystem of the present invention;

FIG. 26 is a front elevation of one flow cytometry system of the presentinvention;

FIG. 27 is an enlarged perspective view of a portion of the system shownin FIG. 26 with parts of the system removed for clarity;

FIG. 28 is a schematic diagram of one embodiment of an epi-illuminationoptics system of the present invention;

FIG. 29 is a perspective view of one embodiment of an epi-illuminationoptics system of the present invention;

FIG. 30 is a side view of the epi-illumination optics system shown inFIG. 29;

FIG. 31 is a top view of the epi-illumination optics system shown inFIGS. 29 and 30;

FIG. 32 is a sectional view of the epi-illumination optics system alongthe plane 32-32 of FIG. 30;

FIG. 33 is a sectional view of a portion of the epi-illumination opticssystem along the plane 33-33 of FIG. 31;

FIG. 34 is a perspective view showing only elements of the opticalfiltering system that are rearward of the dichroic filter of theepi-illumination optics system shown in FIG. 29;

FIG. 35 is a perspective view of another epi-illumination optics systemof the present invention including translational adjustment of thecylindrical lens;

FIG. 36 is a schematic diagram of an interrogation location of oneembodiment of the present invention showing a laser beam focused on afluid stream downstream of the nozzle at a skewed angle of incidence;

FIG. 37 is a schematic diagram of one embodiment of a sort calibrationsystem of the present invention;

FIG. 38 is a schematic diagram of one embodiment of an epi-illuminationsensor for use with the sort calibration shown in FIG. 37;

FIG. 39 is a block diagram of one embodiment of a digital cell analyzer(DCA) and processor controller according to the invention.

FIG. 40 is a schematic diagram of one embodiment of a multi-channelsorter of the present invention showing two channels;

FIG. 41 is a work flow diagram of one embodiment of a multi-channelsorter of the present invention showing four channels;

FIG. 42 is block diagram of one embodiment of an analog cell analyzer(ACA) according to the invention;

FIG. 43 is a graph illustrating a stream of waveform pulses from aphotodetector output detecting fluorescent pulses from cells streamingat an average rate of 10,000 cells/second;

FIG. 44 is an exploded view of FIG. 43 illustrating the stream from aphotodetector output detecting three fluorescent pulses from three cellsstreaming at an average rate of 10,000 cells/second; a square wave of a100 MHz droplet clock has been superimposed on the illustration to showthe synchronization between the three pulses and the square wave pulsesof the droplet clock;

FIGS. 45-48 illustrate movement of a sperm cell relative to a laser beamspot having a narrow width;

FIG. 49 is an exemplary illustration of the digital informationcorresponding to a time-varying analog output from a photodetectordetecting a single fluorescence pulse based on 122 samples at a 105 MHzcontinuous sampling rate;

FIG. 50 is a schematic diagram illustrating the timing relationshipbetween laser pulses, fluorescence emissions from a cell resulting fromthe laser pulses and the digital samples of the photodetector output inone embodiment of the invention;

FIG. 51 is a schematic diagram illustrating how the digital samplesshown in FIG. 50 form a pulse waveform;

FIG. 52 is a schematic diagram of a pulse waveform from an X sperm cellsynchronized with the pulse waveform of a Y sperm cell showing higherpeak intensity in the pulse waveform for the X sperm cell;

FIG. 53 is a schematic diagram of a pulse waveform showing a thresholdand integration window that can be used for pulse analysis;

FIG. 54 is a histogram of a sample containing X and Y sperm cellsshowing the high resolution attainable with slit scanning techniques;

FIG. 55 is histogram of a sample containing X and Y sperm cells showingthe relatively poor resolution attained with standard illumination;

FIGS. 56-59 show fluorescence histograms and scatter plots of peak vs.area for sperm nuclei and live sperm cells;

FIGS. 60-61 illustrate a four-component model of a fluorescenceintensity histogram for sperm cells-FIG. 60 shows raw data and FIG. 61shows model curves generated by one embodiment of an iterative algorithmof the present invention based on the data shown in FIG. 60;

FIGS. 62-63 illustrate a three-component model of a fluorescenceintensity histogram for sperm cells-FIG. 62 shows raw data and FIG. 63shows model curves generated by one embodiment of an iterative algorithmof the present invention based on the data shown in FIG. 62;

FIG. 64 illustrates the non-linear nature of the CSD feature; the toppanel shows average M plots for X-bearing and Y-bearing sperm cells; themiddle panel shows a graph of the first derivatives of these average Mplots (i.e. M′) for signal amplitude values less than the peak height ofthe average Y-bearing fluorescence emission pulse; and the bottom panelshows the difference between the first derivatives (M′_(x)-M′_(y)) as afunction of signal amplitude;

FIG. 65 illustrates one embodiment in which the CSD feature is thecomputed slope of a line that passes through two points on thefluorescence emission pulse;

FIGS. 66-69 illustrate improved discrimination achieved by use of CSDfeature extraction;

FIG. 70 illustrates a bi-variate sort region set on a scatter plot ofCSD vs. pulse area scatter;

FIG. 71 illustrates one embodiment of flow cytometry re-analyses for atest in which the left panel corresponds to the high recovery/coincidentaccept sort strategy (no coincidence abort strategy) and the right panelcorresponds to the high purity/coincident reject sort strategy(coincident abort strategy);

FIG. 72 is a work flow diagram of one embodiment of digital signalprocessing of the present invention;

FIG. 73 is an example of a k-Means clustering strategy that may beemployed according to one embodiment of the present invention;

FIG. 74 is a conceptual illustration and graphical representation ofapplication of a Bayes Minimum Error decision rule to pulse feature dataas may be employed according to one embodiment of the present invention;

FIG. 75 is graphical representation of results obtained using a BayesMinimum Error decision rule and Mahalonobis distance thresh holding asmay be employed according to one embodiment of the present invention;

FIG. 76 is a conceptual illustration of moving window statistics toprovide “forgetting” as may be employed according to one embodiment ofthe present invention;

FIG. 77 is a graphical representation drift compensation as may beemployed according to one embodiment of the present invention;

FIG. 78 illustrates a fluid stream containing an exemplary distributionof particles;

FIG. 79 is a graph showing purity as a function of fluid delivery ratewith a coincident accept sort strategy;

FIG. 80 is a graph showing the percentage of desired particlessuccessfully sorted into the usable population as a function of fluiddelivery rate with a coincident reject sort strategy;

FIG. 81 is a graph showing the inverse relationship between thepercentage of coincident droplets accepted for sorting into a populationof desired particles compared to the percentage of coincident dropletsrejected for sorting into that population;

FIG. 82 is a decision flow diagram showing the overall operation of oneembodiment of a sorting apparatus of the present invention;

FIG. 83 is a side elevation of a cytometer oriented to produce a streamof droplets having a horizontal velocity component and a collectionsystem to collect the droplets;

FIG. 84 is an enlarged perspective view of the collection system shownin FIG. 83 shown relative to the nozzle system and deflector plates;

FIG. 85 is a schematic diagram of one embodiment of a collection systemof the present invention;

FIG. 86 is a front elevation of an intercepting device of the collectionsystem shown in FIG. 83;

FIG. 87 is a side elevation of an intercepting device of the collectionsystem shown in FIG. 83;

FIGS. 88-95 show graphical results of several sperm centrifugationexperiments;

FIGS. 96-98 are schematic diagrams illustrating the steps in oneembodiment of a filtration method of the present invention;

FIG. 99 is a schematic diagram of one embodiment of a filtration systemused to filter sperm cells;

FIG. 100 is a schematic diagram of another filtration system used tofilter sperm cells;

FIGS. 101 and 102 show graphical results of sperm cell filtrationexperiments;

FIG. 103 is a work flow diagram for one embodiment of a cryopreservationmethod of the present invention;

FIG. 104 shows graphical results for a sperm cell cryopreservationexperiment;

FIG. 105 is a work flow diagram for one embodiment of a method ofprocessing sperm cells according to the present invention;

FIG. 106 is a perspective view of one embodiment of a multi-channelparticle sorter of the present invention with parts broken away to showinternal features of the sorter;

FIG. 107 is a perspective view of a manifold system that may be used forfluid delivery in the multi-channel particle sorter of FIG. 106;

FIG. 108 is a perspective view of the manifold system of FIG. 107showing internal fluid connections of the manifold system;

FIG. 109 is a perspective view of the particle sorter shown in FIG. 106with additional elements removed or partially removed to better showinternal features of the sorter;

FIG. 110 is a front elevation of the particle sorter shown in FIG. 106;

FIG. 111 is a side elevation of the particle sorter shown in FIG. 106with the side wall of the housing removed to show internal features ofthe sorter;

FIG. 112 is a side elevation of the particle sorter shown in FIG. 106(taken from the side opposite the side from which FIG. 107 was taken)with the side wall removed to show internal features of the sorter;

FIG. 113 is a perspective view of the particle sorter shown in FIG. 106taken from an angle behind the sorter and with the back cover removed toshow internal features of the sorter;

FIG. 114 is a perspective view of a portion of the particle sorter shownin FIG. 106 showing the mounting of multiple nozzle systems to a crossbar;

FIG. 115 is a perspective view of a portion of the particle sorter shownin FIG. 106 showing the relative positions of the collection system andother parts of the particle sorter;

FIG. 116 is a schematic diagram of one embodiment of a fluid deliverysystem for a multi-channel sorter of the present invention;

FIGS. 117 and 118 are schematic diagrams of two different laserbeamsplitting systems;

FIGS. 119 and 120 are perspective views of another multi-channel systemof the present invention;

FIGS. 121-134 show graphical results of various experiments;

FIG. 135 is a schematic diagram of one alternative embodiment for anozzle system of the present invention wherein the nozzle directs thefluid stream through a capillary tube;

FIG. 136 is a schematic diagram of one embodiment of a photo damagesorting system of the present invention;

FIG. 137 is a schematic diagram of an alternative sorting system basedon fluidic switching that may be used in an apparatus employing thetechnology of the present invention; and

FIG. 138 is a schematic diagram of an alternative sorting system basedon a high-speed droplet interference stream that diverts selecteddiscrete segments of the fluid stream carrying the analyzed particles.

Corresponding parts are designated by corresponding reference numbersthroughout the drawings. A parts list with associated reference numeralsfor each part follows. The parts list is provided with section headingsgenerally corresponding to section headings in the specification tofacilitate use of the parts list. Generally, each section of the partslist provides a reference numeral for the parts that are introduced forthe first time in the corresponding section of the Detailed Description.

PARTS LIST WITH ASSOCIATED REFERENCE NUMERALS FOR EACH PART

General Overview

-   39 Semen Collection-   41 Label Semen-   41A Add Buffer-   43 Quality Control-   47 Washing-   48 Staining Fluid-   49 Staining-   51 Incubation-   53 Load into Sample Introduction Device of Flow Cytometer-   54 Add Sheath Fluid Through Flow Cytometry-   55 Sorting-   57 Collecting Sorted Sperm-   58A Add Collection Fluid-   58B Concentrate Sperm Cells-   58C Add Cryoextender-   59 Load Sorted Sperm into Straws-   61 Cryopreservation-   63 Packing in Liquid Nitrogen-   65 Distribution-   67 Sales-   69 Storage-   71 Artificial Insemination

Flow Cytometry

-   1 System (Overall)-   3 Supply of Carrier Fluid-   7 Supply of Sheath Fluid-   9 Flow Cytometry Apparatus Having Sorting Capabilities-   15 Fluid Delivery System-   17 Carrier Fluid-   19 Sheath Fluid-   21 Stream of Fluid-   23 Stream of Particles-   25 Beam of Electromagnetic Radiation-   31 Electromagnetic Radiation Emission from Particles-   33 Droplets-   35 Particles Contained in Droplets

Flow Cytometry Apparatus (Single Channel)

-   101 Nozzle System-   103 Nozzle Orifice-   105 Transducer-   107 Droplet Break-off-   109 Optics System-   115 Interrogation Location-   117 Photodetector-   119 Sorting System-   123 First Different Group or Population of Droplets-   125 Second Different Group or Population of Droplets-   2201 Collection System-   131 Processor

Nozzle System

-   133 Cylindrical Flow Body-   135 Central Longitudinal Bore-   137 Nozzle-   139 Funnel-shaped Nozzle Body-   141 Passage Through Nozzle Body-   145 Internally Threaded Counterbore-   149 Threaded Projection or Stud-   155 O-ring Seal-   157 Conduit (Tubular Needle)-   167 Annular Space (Gap)-   173 Radial Bore in Flow Body (Sheath Fluid)-   183 Second Radial Bore (Additional Sheath Fluid)-   189 Central Core of Carrier Fluid-   191 Outer Co-axial Sheath of Fluid

Cell Orientation

-   201 Bovine Sperm Cell-   205 Paddle-shaped Head-   207 Flat Wide Opposite Faces-   209 Narrow Edges-   211 Sperm Equator-   213 Nucleus-   215 Tail-   217 Nucleus Length-   219 Head Length-   221 Head Width-   223 Overall Length-   225 Localized Region Within Nucleus-   227 Direction of Stream Flow-   229 Angular Envelope in Which Light Beam Strikes Wide Face-   R1 Angular Range-   P Plane

Nozzle Design

-   231 Interior of Nozzle Body-   233 Interior Surface of Nozzle Body-   235 First Axially Tapered Region-   237 Second Axially Tapered Region-   239 Third Axially Tapered Region-   247 Longitudinal Axis of Nozzle-   249 Fourth Region Interior of Nozzle-   251 Axial Length of Fourth Region-   255 Orifice Member-   257 Counterbore at Front End of Nozzle-   259 First Torsional Zone-   261 Second Torsional Zone-   263 Surface of First Torsional Zone-   267 Surface of Second Torsional Zone-   271 Torsional Forces-   273 Axial Length of First Torsional Zone-   275 Axial Length of First Tapered Region-   277 Axial Length of Second Tapered Region-   279 Axial Length of Second Torsional Zone-   309 Conical Upstream Surface of Orifice Member-   315 Cylindrical Downstream Surface of Orifice Member-   317 Axial Length of Conical Upstream Surface-   327 Axial Length of Downstream Surface

Orienting Baffle

-   2001 Orienting Baffle-   2003 Baffle Plate-   2005 Baffle Holder-   2007 Upstream Leg-   2009 Downstream Leg-   2015 Line of Intersection-   2017 Central Axis of Nozzle Body-   2019 Curved Edge of Upstream Leg-   2025 Distance Lower Leg Extends Downstream-   2027 Overall Length of Baffle Holder-   2029 Exterior Diameter of Baffle Holder-   2031 Interior Diameter of Baffle Holder-   2033 Distance Between Line of Intersection and Center of Nozzle-   2035 Upstream End of Baffle-   2037 Inclined Surface of Baffle Holder-   2039 Side Edges of Downstream Leg-   2041 Downstream Edge of Downstream Leg-   2049 Gap Between Baffle Plate and Baffle Holder-   2051 Inside surface of Baffle Holder-   2053 Volume Behind Baffle Plate-   2055 Interior Volume of Nozzle-   2057 Longitudinal Axis of Cylindrical Baffle Holder-   2059 Line Through Major Axis of Ellipse D-   2061 Distance Between Injection Needle and Baffle-   2067 Downstream End of Baffle Holder-   2069 Contact Points Between Baffle Holder and Nozzle-   2071 O-Rings-   2077 Downstream End of Nozzle Holder (Boss)-   2079 Interior Diameter of Boss-   2081 Portion of Sheath Fluid Between Core Stream and Nozzle Surface-   2087 Cross Section Upstream (A)-   2089 Cross Section at Baffle (B)-   2091 Cross Section at Baffle (C)-   2093 Cross Section at Baffle (D)-   2094 Cross Section Downstream of Baffle (E)-   2097 Perpendicular Baffle System-   2095 Air Bubble-   2099 Perpendicular Baffle Plate-   2101 Curved Edge of Perpendicular Baffle Plate-   2103 Straight Edge of Perpendicular Baffle Plate-   2105 O-ring-   2107 Annular Shoulder (Shelf) in Nozzle-   2109 Outer Diameter of Sample Injection Needle (Conduit)-   2151 Nozzle System Having an Offset Sample Introduction Conduit

Nozzle Mounting and Adjustment

-   331 Nozzle Mount-   333 First Linear Stage-   337 Second Linear Stage-   339 X Axis-   341 Y Axis-   343 Third Rotational Stage-   345 Z Axis-   347 Fixed First Stage Member (Not Shown)-   349 Frame for First Fixed Stage Member-   355 Movable First Stage Member-   357 Actuator (Micrometer) for First Stage-   359 Fixed Second Stage Member-   361 Movable Second Stage Member-   363 Actuator (Micrometer) for Second Stage-   365 Fixed Third Stage Member-   371 Movable Third Stage Member-   373 Actuator (Micrometer) for Third Stage-   375 Generally Upward Direction of Stream Containing Cells-   377 Angle of Upward Direction

Transducer and Droplet Formation

-   379 Collar-   381 Piezoelectric Element (Not Shown)-   383 Terminals-   D Diameter of Stream

Break-Off Sensor

-   389 Break-off Sensor-   391 Microprocessor-   393 Light Source-   395 Linear Photoarray (Photodiodes)-   401 Lens for Droplet Break-off Sensor-   405 Current to Voltage Op-amp Circuits-   407 Track/hold Amplifiers-   409 Sinewave Generator (Track/hold Signal)-   411 A/D Converter-   412 Camera System-   413 Strobe-   414A Mask-   414B Slit-Shaped Opening in Mask

Epi-Illumination Optics System

-   415 Epi-illumination System-   417 Epi-illumination Instrument-   419 Longitudinal Optical Axis-   425 Beam Spot-   427 Axis of Focused Illumination Beam-   429 Rectangular Base-   431 Reflecting Filter-   435 Laser or Arc-lamp-   437 Conditioning Lens Assembly-   439 Opening in-   441 Side Wall of a Dichroic Chamber-   443 Dichroic Chamber-   445 Retaining Ring-   447 Neutral Density Filter-   449 Cylindrical Lens-   455 Lens Holder-   457 Jam Nut-   459 Elliptical Cross Section of Beam Spot-   461 Clips for Reflecting Filter-   463 Filter Holder-   465 Angular Face of Filter Holder-   467 Openings in Filter Holder-   469 Linear Stage for Filter Holder-   471 X Axis-   473 Outrigger-   475 Actuator for Linear Stage-   477 Dichroic Filter-   479 Clips for Dichroic Filter-   485 Frame for Dichroic Filter-   487 Forward Direction-   489 Longitudinal Optical Axis of the Optical Instrument-   491 Focusing Lens Assembly-   497 Fluorescent Pulse Waveform or Signal Emitted by Cell-   498 Excitation Spatial Function-   501 Microscope Adapter-   503 Opening in Front Wall of Dichroic Chamber-   505 Front Wall of Dichroic Chamber-   507 Focusing Barrel-   509 Lens Mount Barrels-   511 Focusing Lens-   513 Rearward Direction-   515 Telescoping Focus Adjustment-   517 Collimated Emitted Light-   519 Filtering System-   521 Emission Filter-   523 Emission Filter Holder-   525 Opening in Back Wall of Dichroic Chamber-   527 Back Wall of Dichroic Chamber-   529 Alignment Pellicle Assembly-   531 Slider of Alignment Pellicle-   533 Rail for Filter Assembly Components-   535 Filter Holder for Alignment Pellicle-   539 Pellicle Filter Element-   541 Clips for Securing Filter Element to Filter Holder-   543 Angle for Alignment Pellicle Relative to Optical Axis-   545 Fasteners for Securing Slider to Base-   547 Parallel Slots in Base-   549 Aspheric Lens-   551 Holder for Aspheric Lens-   553 Frame for Aspheric Lens-   557 Fasteners for Aspheric Lens-   559 Spatial Filter-   561 Aperture Plates-   563 Frame for Spatial Filter Plates-   567 Vertical Slit-   571 Horizontal Slit-   573 Aperture-   575 Vertical Dimension-   577 Horizontal Dimension-   579 Collection Volume-   583 Plate Holder-   587 Fasteners for Plate Holder-   589 Backing Member for Aperture Plates-   449A Adjustable Mounting Assembly-   449B Slots-   449C Slots-   450 Epi-illumination that Reflects Fluorescence Emissions-   451 Dichroic Filter

Photodetector

-   591 Mounting Plate for Photodetector-   595 Fasteners for Photodetector

Angle of Beam Incidence

-   605 Distance Between Interrogation Location and Nozzle Orifice-   609 Beam Axis-   A Angle of Incidence

Focused Beam Spot

-   L1 Length along Major Axis-   W1 Width along Minor Axis

Sorting System

-   627 Charging Device-   629 Charged Deflector Plates-   631 Charging Element-   633 Opening in Charging Element-   635 Power Supply for Deflector Plates-   5001 Adjustable Mounting Assembly-   5003 Mounting Assembly Adjustment Board-   5005 Mounting Assembly Backing-   5007 Fasteners-   5009 Slots-   5011 Translation Axis-   5013 Translation Axis-   5015 Mounting Assembly Adjustment Board-   5017 Fasteners-   5019 Slots-   5021 Fixed Support-   5023 Fasteners-   5025 Spring

Automat Sort Calibration

-   4201 Calibration System-   4203 Epi-illumination Sensor-   4205 Fiber Optic Cable-   4207 Dichroic Filter-   4209 Lens System-   4211 Fluorescent Emission from Particle in Droplet-   4213 Photodetector-   4215 Beam Stop

Sort System Fault Correction

-   5047 Debris Removal System for Charging Element-   5049 Debris Removal System for Deflector Plates-   5051 Support for Charging Element-   5053 Vacuum Passage-   5055 Vacuum Line-   5057 Opening Adjacent Charging Element-   5058 Fitting-   5059 Compressed Gas Line-   5061 Manifold-   5063 Air Passages-   5064 Openings-   5065 Fitting-   5066 Side of Deflector Plate

Protection of Sorted Sample

-   4033 Collection Vessel-   4041 Contamination Prevention Mechanism-   4043 Pneumatic Actuator-   4045 Swing Arm-   4047 End of Swing Arm

Fluid Delivery System

-   645 Syringe Pump-   647 Flow Line from Pump to Carrier Supply-   649 Vessel for Containing Supply of Carrier Fluid-   651 Line from Pump to Injection Needle-   657 Supply Line from Syringe Pump to Needle-   659 Variable Speed Motor-   661 Second Vessel—for Supply of Sheath Fluid-   667 Supply Line for Connecting Sheath Fluid to Radial Bore in Nozzle-   669 Control Valve in Supply Line-   671 Gas Pressure System for Sheath Fluid-   675 Source of Pressurized Gas-   679 Air Line for Pressurized Gas-   681 Regulator for Controlling Pressure Supplied to Sheath Fluid Tank-   683 Two-way Valve in Air Line

Control

-   689 A/D Converter-   693 Relative Beam Intensity Experienced by Point Moving Through Beam    Spot-   695 Relative Emitted Pulse Intensity From Sperm Traversing Beam Spot-   d Distance Between Nozzle and Droplet Break-off Location

Signal Processing

-   701 Output Signal From Photodetector-   703 Droplet Generation Clock Signals-   705 Digital Signal Processing (Digital Cell Analyzer)-   707 Digital Signal from A/D-   735 PC/Computer Terminal-   737 Master Clock (128×Clock Signal)-   739 Data Acquisition (HH1′)-   741 Initializing Detection Parameters (HH1)-   745 Initializing Discrimination Parameters (HH2)-   747 Digital Pulse Detection (HH3)-   749 Digital Pulse Analysis—Feature Extraction (HH4)-   753 Pulse Area (HH5)-   755 Pulse Peak (HH6)-   757 Pulse Discrimination (HH7)-   759 Sorting (HH8)-   761 Drift Analysis (HH9)-   763 Decision Boundary for Bayes Rules-   769 Initialize-   771 System Check-   773 User Interaction-   775 Retry (Up to Three Times)-   777 Flush-   779 Bead Quality Control-   781 Aspirate Sample-   783 Sample Quality Control-   785 Start Sample-   787 Sort On-   789 Sample Complete-   791 Continue Sample-   793 Sort Off-   795 X/Y Discrimination Optimum-   797 Set X/Y Discrimination-   799 Discrimination OK-   801 Rate Optimum-   803 Set Syringe Rate-   805 Rate OK-   807 System Check-   809 System Reset-   811 System OK-   813 Exemplary Overall Operational Flow-   825 Integrator-   827 Width/Area Comparator-   829 Dynamic Threshold Calculator-   831 Pulse Discrimination-   833 JTAG Port I/O-   837 Window Comparator (Area)-   839 Pulse Width and Trigger Logic-   841 Sort Decision-   843 I/O Controllers-   845 Slave Controllers-   847 Sort Controller Board-   849 USB-   851 DSP Board SDRAM-   853 Sort Signal-   854 Low-Pass Filter-   855 I/O Board SDRAM-   857 Processor I/O-   859 Peripheral I/O Bus-   861 Sort Pulse Generator-   863 Data Management Processor-   865 Pulse Detection Processor-   867 Feature Extraction Processor-   873 Sort Processor-   875 DSP Board RAM-   OL Inverse Relationship Between Coincident Droplets in Usable    Population Compared to Coincident Droplets in Unusable Population-   P1 Point on Line OL Corresponding to 85% Purity-   LL Point on Line OL Corresponding to 60% Collection of Desired    Particles-   OR Operating Range (Segment of OL Between P1 and LL)-   6000 Raw Data-   6001 1st Population of Non-aligned Cells-   6003 2nd Population of Non-aligned Cells-   6005 Aligned Y Population-   6007 Aligned X Population-   6010 Raw Data-   6011 Population of Non-aligned Cells-   6015 Aligned Y Population-   6017 Aligned X Population

Multi-Channel System

-   1001 Multi-channel System-   1003 Flow Cytometry Units-   1005 Common Particle Supply-   1007 Common Source of Electromagnetic Radiation-   1009 Common Housing-   1011 Common Input for Control-   1019 Common Output-   1021 Common Fluid Delivery System-   1023 Common Temperature Control System-   1025 Common Power Source-   1027 Common Waste Recovery System-   1029 Common Deflector Plate System-   1031 Common Cleaning System

Common Housing

-   1069 Base-   1071 Two Side Walls-   1073 Lower Pair of Shoulders-   1075 Lower Cover Panel-   1077 Front of Housing-   1081 Upper Pair of Shoulders-   1083 Upper Cover Panel-   1085 Rear of Housing-   1087 Framework for Mounting Multiple Cytometry Units-   1089 Cross Bar Affixed to Side Walls of Housing (For Attaching    Nozzle Mounts)-   1093 Angled Mounting Plate Extending Between Side Walls

Common Fluid Supply

-   1105 Pump for Carrier Fluid-   1107 Common Supply of Carrier Fluid-   1115 Gas Pressure System for Sheath Fluid-   1117 Common Supply of Sheath Fluid-   1121 Manifold System-   1123 Vessel Containing Common Supply of Carrier Fluid-   1125 Holder for Vessel-   1133 Holding Block-   1135 Cavity for Receiving Vessel-   1137 Second Cavity for Buffer Material-   1139 Vessel for Buffer Material-   1141 Syringe Pump-   1147 Supply Line from Syringe Pump to Manifold-   1149 Three-way Valve Controlling Carrier and Buffer Fluid-   1155 Vessel for Common Supply of Sheath Fluid-   1157 Supply Line from Sheath Fluid Vessel to Manifold-   1161 Source of Pressurized Gas-   1163 Gas Line-   1165 Regulator in Gas Line-   1167 Two-way Valve for Gas Line Between Gas Source and Sheath Fluid    Tank-   1169 Gas Line for Pressurizing a Supply of Cleaning Solution-   1173 Tank for Cleaning Solution-   1175 Two-way Valve for Gas Line for Cleaning Solution-   1177 Manifold-   1179 Laminated Block-   1181 Passages-   1185 Fluid Flow Circuit-   1189 Inlets Connected to Syringe Pump-   1191 Inlets Connected to Supply of Sheath Fluid-   1193 Outlets for Carrier Fluid and Sheath Fluid-   V1-V6 Valves for Controlling Flow Through Manifold Passages-   1203 Frame Member (For Attaching Manifold Block)-   1205 Fittings Threaded into Block-   1207 Sample Reservoir-   V1A-V1D Two-way Valves (For Controlling Flow of Sample Fluid to    Nozzles)-   1217 Needle of Sample Reservoir-   1221 Waste System-   1223 Waste Tank (Receptacle)-   1225 Mechanism Such as Vacuum Pump (For Generating Vacuum)-   1227 Waste Lines (Connecting Valves V1A-V1D to Waste Tank)-   1233 Hydrophobic Filter (In Line Connecting Waste Tank and Vacuum    Pump)-   1235 Fluid Circuit for Sheath Fluid-   V2A-V2D Two-Way Valves (For Controlling Flow of Sheath Fluid to    Nozzles)-   1241 Sheath Supply Line-   1247 Waste Lines Connecting Sheath Fluid Flow Circuitry to Waste    Tank

Common Power Supply and Controls

-   1249 Common Power Supply-   1251 Common Power Delivery Systems-   1253 Common Input (GUI)-   1255 Common Output (To Microprocessor)

Common Temperature Control

-   1257 Temperature Control System-   1259 Fluid Flow Circuit (For Temperature Control)-   1263 Fluid Passages (For Temperature Control in Holding Block)-   1265 Control Unit-   1269 Fluid Passages (For Temperature Control in Manifold)-   V6 Shut off Valve

Common Light Beam and Beam Splitting System

-   1270 Beam splitter-   1270A First Beam from Beam splitter-   1270B Second Beam from Beam splitter-   1271 Second Beam splitter-   1271A First Beam from Second Beam splitter-   1271B Second Beam from Second Beam splitter-   1272 Third Beam splitter-   1272A First Beam from Third Beam Splitter-   1272B Second Beam from Third Beam splitter-   1273 Beam Guidance System-   1279 Lower Filter Assembly-   1281 Upper Mirror Assembly-   1285 Base (For Lower Filter Assembly)-   1289 Stage (For Lower Filter Assembly)-   1291 Mechanism for Moving Stage (Micrometer)-   1293 Tiltable Platform on the Stage-   1295 Mirror (On Platform)-   1297 Base (For Upper Mirror Assembly)-   1299 Stage (For Upper Mirror Assembly)-   1301 Tiltable Platform (For Upper Mirror Assembly)-   1303 Mirror (For Upper Mirror Assembly)-   1305 Mechanism for Moving Upper Stage-   1309 Target Plates (Affixed to Side Wall of Housing)-   1311 Vertically Aligned Holes (In the Target Plates)-   1315 1st Reflecting Filter-   1317 2nd Reflecting Filter-   1319 3rd Reflecting Filter-   1321 4th Reflecting Filter

Common Deflector Plates

-   1331 Two Common Deflector Plates-   1333 Frame (For Mounting Common Deflector Plates on Housing)

Modular Multi-Channel System

-   4001 Multi-Channel System-   4009 Modular Cytometry Unit-   4011 Housing for Modular Unit-   4013 Common Housing-   4015 Laser-   4017 Beam Splitting and Guidance System-   4021 Hole for Laser to Enter Modular Housing-   4023 Plate to Cover Exit Hole-   4025 Collection System for System

Capillary Tube Nozzle System

-   1335 Capillary Tube Nozzle System-   1337 Capillary Tube-   1341 Chamber Filled with Light-transmitting Medium

Alternative Sorting Systems

-   1351 Photodamage Sorting System-   1353 Second Laser-   1355 Collection Receptacle-   1357 Fluid Switching System-   1359 Fluid Switching Device-   1361 Capillary Branch to First Collection Vessel-   1365 Capillary Branch to Second Collection Vessel-   1367 Transducer (For Creating Pressure Waves for Selectively    Controlling Direction of Fluid Flow)-   1369 Capillary Tube on End of Nozzle-   1371 Droplet Interference Stream Sorting System-   1373 High-Speed Droplet Interference Stream-   1375 Droplet Generation System for High-Speed Droplet Stream-   1377 High-Speed Nozzle System-   1379 High-Speed Fluid Stream-   1381 Transducer for Droplet Interference Stream Generation-   1383 High-Speed Droplets-   1387 Electric Deflection Plate for High-Speed Droplet Deflection-   1389 Uncharged Droplets-   1391 Charged Droplets-   1397 Diverted Segment of Fluid Stream-   1399 Intersection of High-Speed Droplet Stream with Coaxial Fluid    Stream-   1403 Collection Capillaries

Collection System

-   2201 Collection System-   2203 Intercepting Device-   2205 Impact Surface-   2207 Collection Vessel-   2211 Droplet Entryway-   2213 Bulb of Pipette-   2215 Pipette-   2217 Inside Wall of Pipette-   2225 Guide Tube-   2227 Collection System Frame-   2229 Circular Holder-   2231 Set Screw for Intercepting Device Height-   2233 Mounting Plate-   2235 Set Screws for Lateral Adjustment-   2241 Lateral Slot-   2243 Tray for Holding Collection Vessels-   2245 Exit Window-   2247 First Intercepting Device-   2249 Second Intercepting Device-   2265 Stray Droplets

Collection Fluid

-   2301 Collection Fluid

Filtration

-   2401 Filter-   2403 Collection Vessel for Filtration-   2405 Concentrated Slurry Containing Sperm Cells-   2409 Syringe Mechanism-   2411 Cannula Filter-   2413 Resuspension fluid-   2419 Second Container-   2421 Syringe for Filtration Experiment-   2423 Sample for Filtration Experiment-   2425 Filter for Filtration Experiment-   2427 Vacuum Pump for Filtration Experiment-   2431 Syringe for Filtration Experiment II-   2433 Sample for Filtration Experiment II-   2435 Filter for Filtration Experiment II-   2437 Filter Holder for Filtration Experiment II

Cryopreservation

-   2501 Adjust Concentration-   2503 Add Cryoprotectant-   2505 Add Protein Source-   2507 Load in Straws-   2509 Cool to Holding Temperature-   2511 Maintain at Holding Temperature-   2513 Cool to Temperature Approaching Critical Zone-   2515 Cool Through Range of Ice Crystal Formation-   2517 Immerse in Liquid Nitrogen

Common Collection System

-   2801 Common Collection System-   2803 Common Frame for Intercepting Devices-   2805 Waste Trough-   2807 Tray for Collection Vessels

Pulsed Laser System

-   3001 Pulsed Laser-   3003 Laser Pulse Sensor-   3005 Laser Pulse-   3007 Fluorescence Pulse Lifetime Decay-   3009 Digital Sample

DETAILED DESCRIPTION OF EMBODIMENTS

The embodiments described below relate to collection and processing ofanimal semen, particularly to processing semen from a domestic animal tosort the sperm cells according to a specified DNA characteristic (e.g.,X/Y chromosome content to preselect the gender of offspring). A numberof inventive technologies are combined to achieve the results describedbelow. However, it will be understood that the inventive technologiesdescribed herein may be applied to other applications without deviatingfrom the scope of this invention.

General Overview

FIG. 1 is a work flow diagram providing an overview of the steps in oneexemplary process of the present invention. The process starts withcollection of neat semen samples from one or more male animals (e.g.,bulls) at step 39. The semen samples are labeled for identification atstep 41, contacted with a buffer, at step 41A and transported to aprocessing facility. In addition to the buffer, additives may also beadded at step 41A, including, for example, an energy source, a proteinsource, an antibiotic, and/or a composition which regulatesoxidation/reduction reactions intracellularly and/or extracellularly. Anoptional quality control test may be performed at step 43 to insure thatthe quality of each sample (e.g., sperm motility) is sufficient toindicate that the final product is likely to meet minimal qualitycriteria. An optional washing step may be performed at step 47. At step47A the staining protocol that will be used for processing is selectedby using various staining protocols to stain aliquots of the sample andthen analyzing the sortability of each aliquot to identify a desiredstaining protocol for that particular sample. Staining according to theselected staining protocol is performed at step 49 by adding a stainingfluid 48 containing a chemical dye (e.g., a DNA selective fluorescentdye) to each sample. In addition to the staining fluid, additives mayalso be added at step 48, including, for example, an energy source, aprotein source, an antibiotic, and/or a composition which regulatesoxidation/reduction reactions intracellularly and/or extracellularly.The samples are incubated at step 51 to allow for uptake of the dye bythe sperm. Then a sample is loaded into the sample introduction deviceof a flow cytometer at step 53. The sample fluid is introduced into theflow cytometer along with a sheath fluid at step 54. In addition to thesheath fluid, additives may also be added at step 54, including, forexample, an energy source, a protein source, an antibiotic, and/or acomposition which regulates oxidation/reduction reactionsintracellularly and/or extracellularly. At step 55 the flow cytometersorts the sperm cells according to a specified DNA characteristic, aswill be described below. As the sorted sperm cells are collected by thecollection system of the flow cytometer at step 57, they are added to acollection vessel that contains a collection fluid or cryoextender atstep 58A. In addition to the collection fluid, additives may also beadded at step 58A, including, for example, an energy source, a proteinsource, an antibiotic, and/or a composition which regulatesoxidation/reduction reactions intracellularly and/or extracellularly. Bythis time the sperm cells are in a solution that has been diluted by thevarious fluids added throughout the process. Accordingly, the populationof sperm cells having the desired DNA characteristic are concentrated atstep 58B for use in commercial artificial insemination. A cryoextenderis added to the concentrated sorted sperm cells at step 58C. In additionto the cryoextender, additives may also be added at step 58C, including,for example, an energy source, a protein source, an antibiotic, and/or acomposition which regulates oxidation/reduction reactionsintracellularly and/or extracellularly. The sperm cells are then packedin tubular containers (referred to in the breeding industry as “straws”)at step 59 and cryopreserved at step 61. The cryopreserved sperm arepacked for storage in liquid nitrogen at step 63. The cryopreservedsperm are then distributed through a commercial distribution system atstep 65 and sold to animal breeders at step 67. The animal breeders maystore the cryopreserved sperm at step 69 until they are ready to use thesperm to artificially inseminate a female animal (e.g., cow) at step 71.As will be discussed below, one embodiment of the present inventioninvolves temperature control through substantially the entire process.Likewise, completion of the various steps within defined time limits isone aspect of another embodiment of the present invention. This overallprocess is only one example of how the present invention can be used,and it will be understood that some of the aforementioned steps can bedeleted and/or others added. The sorted sperm cells can also be used formicroinjection or other in vitro fertilization, followed by embryotransplant into a recipient female animal.

The steps of the overall process incorporating advances of the presentinvention are described in detail below. While a particular processdescribed is in the context of sorting animal sperm (e.g., bovinesperm), it will be understood that the various aspects of this inventionare more generally applicable to any type of sperm (equine, porcine, andothers), even more generally to any type of cells, and even moregenerally to any type of particles, organic and inorganic, includinglatex particles, magnetic particles, chromosomes, sub-cellular elements,protoplasts, and starch particles. These particles generally fall withina size range of 0.5 to 200 microns, but the technology of this inventionis not limited to this range.

Sample Collection and Dilution Sample Collection

The sperm sample to be sorted may be a freshly collected sample from asource animal, such as bovine, equine, porcine, or other mammaliansource, or a thawed, previously cryopreserved sample. Moreover, thesample may be a single ejaculate, multiple pooled ejaculates from thesame mammal, or multiple pooled ejaculates from two or more animals.

Various collection methods are known and include the gloved-hand method,use of an artificial vagina, and electro-ejaculation. The sperm arepreferably collected or quickly transferred into an insulated containerto avoid a rapid temperature change from physiological temperatures(typically about 35° C. to about 39° C.). The ejaculate typicallycontains about 0.5 to 15 billion sperm per milliliter, depending uponthe species and particular animal.

Regardless of the method of collection, an aliquot may be drawn from thesperm sample and evaluated for various characteristics, such as forexample, sperm concentration, sperm motility, sperm progressivemotility, sample pH, sperm membrane integrity, and sperm morphology.This data may be obtained by examination of the sperm using, forexample, the Hamilton-Thorn Motility Analyzer (IVOS), according tostandard and well known procedures (see, for example, Farrell et al.Theriogenology (1998) 49 (4): 871-9; and U.S. Pat. Nos. 4,896,966 and4,896,967).

Dilution

The sperm sample may be combined with a buffer (in the form of a solidor solution) to form a sperm suspension. Among other things, the buffermay enhance sperm viability by buffering the suspension againstsignificant changes in pH or osmotic pressure. Generally, a buffer isnon-toxic to the cells and is compatible with the dye used to stain thecells. Exemplary buffers include phosphates, diphosphates, citrates,acetates, lactates, and combinations thereof. Presently preferredbuffers include TCA, TEST, sodium citrate, HEPES, TL, TES, citric acidmonohydrate, HEPEST (Gradipore, St. Louis, Mo.), PBS (Johnson et al.,Gamete Research, 17: 203-212 (1987)), and Dulbecco's PBS (InvitrogenCorp., Carlsbad, Calif.).

One or more buffers may be combined together or with additives asdiscussed below to form a buffered solution, and the buffered solutioncombined with the sperm sample to form a sperm suspension. A bufferedsolution may also contain one or more additives, as described in greaterdetail below. Exemplary buffered solutions are described in Table I.Preferred buffered solutions include a solution comprising 3% TRIS base,2% citric acid monohydrate, and 1% fructose (w/v) in water at a pH ofabout 7.0, a solution designated as TCA #1 in Table 1, and a solutiondesignated as TCA #2 in Table I.

TABLE I Buffered Solutions Na COMPONENTS TCA#1 TCA#2 TEST Citrate HEPESTL Sodium chloride (NaCl)  7.6 g 5.84 g Potassium chloride (KCl)  0.3 g0.23 g Sodium bicarbonate (NaHCO3)  2.1 g Sodium phosphate monobasic0.04 g (NaH2PO4—H2O) (+)-2-hydroxyproprionic 3.68 ml acid (Na Lactate)Magnesium chloride (MgCl2)  0.1 g 0.08 g N-(2-hydroxyethyl) 2.38 g 2.38g piperazine-N′-(2- ethansulfonic acid) (HEPES) tris(hydroxymethyl) 30.3 g 32.02 g 10.28 g amimonethane (TRIS base) Citric Acid Monohydrate15.75 g 18.68 g Na Citrate Dihydrate 29 g 2-[(2-hydroxy-1,1- 43.25 gbis[hyroxymethyl] ethyl) aminoethanesulfonic acid (TES) Fructose  12.5 g 2.67 g 10 g 2.52 g D-Glucose    2 g Steptamycin  0.25 g Penicillin-G 0.15 g Water 1 liter 1 liter 1 liter 1 liter 1 liter 1 liter Target pH7.35 7.35 7.35 7.35 7.35 7.35 Target osmolality ~314 ~300 ~302 ~316 ~298~296 (milliosmols/kg H2O)

Alternatively, the sperm may be combined with a metabolic inhibitor toform an inhibited sperm suspension. Metabolic inhibitors cause the spermcells to emulate sperm cells of the epididymis of a mammal, such as forexample a bull, by simulating the fluid environment of the epididymis orepididymal tract of the mammal. Such an inhibitor would reduce orinhibit the motility and metabolic activity of the sperm. Exemplaryinhibitors of this class include carbonate based inhibitors, such as forexample those disclosed in Salisbury & Graves, J. Reprod. Fertil., 6:351-359 (1963). A preferred inhibitor of this type comprises NaHCO₃,KHCO₃, and C₆H₈0₇H₂O. A more preferred inhibitor of this type comprises0.204 g NaHCO₃, 0.433 g KHCO₃, and 0.473 g and C₆H₈0₇.H₂O per 25 mL ofpurified water (0.097 moles/L of NaHCO₃, 0.173 moles/L of KHC0₃, 0.090moles/L and C₆H₈0₇.H₂O in water).

In addition to a buffer, the sperm suspension may also contain a rangeof additives to enhance sperm viability or motility. Exemplary additivesinclude energy sources, protein sources, antibiotics, and compositionswhich regulate oxidation/reduction reactions intracellularly and/orextracellularly. One or more of these additives may be introduced intothe buffer or buffered solution before the formation of the spermsuspension or, alternatively, may be separately introduced into thesperm suspension.

One or more energy sources may be added to minimize or inhibit the spermcells from oxidizing intracellular phospholipids and other cellularcomponents. Exemplary energy sources include monosaccharides, such asfructose, glucose, galactose and mannose, and disaccharides, such assucrose, lactose, maltose, and trehalose, as well as otherpolysaccharides. For example, the resulting sperm suspension may includeabout 1% (w/v) to about 4% (w/v) of the energy source(s). If included,the energy source is preferably fructose and the sperm suspensioncontains about 2.5% (w/v).

To minimize dilution shock, provide support to the cells, or dispersethe cells throughout the suspension, a protein source may also beincluded in the buffer, buffered solution, or sperm suspension.Exemplary protein sources include egg yolk, egg yolk extract, milk(including heat homogenized and skim), milk extract, soy protein, soyprotein extract, serum albumin, bovine serum albumin, human serumsubstitute supplement, and combinations thereof. Albumin, and moreparticularly bovine serum albumin (BSA), is a preferred protein source.For example, if included, BSA may be present in the sperm suspension inan amount of less than about 5.0% (w/v), preferably less than about 2%(w/v), more preferably less than about 1% (w/v), and most preferably inan amount of about 0.1% (w/v).

The use of a protein source, such BSA, alone may initiate the process ofcapacitation in a percentage of the sperm cells in the suspension. It ispreferred that this process take place in the female reproductive tract.Therefore, in order to inhibit the initiation of capacitation duringdilution, as well as during the subsequent staining and sorting, analternative protein source or a protein substitute may be included inthe sperm suspension. The alternative protein source or proteinsubstitute possess the advantageous effects of a typical protein source,such as BSA, in addition to the ability to inhibit the initiation ofcapacitation in a larger percentage of the cells in the spermsuspension. Examples of alternative protein sources include human serumsubstitute supplement (SSS) (Irvine Scientific, Santa Ana, Calif.) andcholesterol enhancer BSA, while an example of a protein substituteincludes a polyvinyl alcohol, such as for example, a low to mediumviscosity polyvinyl alcohol generally of a molecular weight of about30,000 to about 60,000. Generally, if included, these compositions willbe present in the same amounts as disclosed above with respect to BSA,with the total albumin content of the buffer or buffered solutiongenerally not exceeding about 5.0% (w/v).

An antibiotic may be added to the sperm suspension in order to inhibitbacterial growth. Exemplary antibiotics include, for example, tylosin,gentamicin, lincomycin, spectinomycin, Linco-Spectin® (lincomycinhydrochloride-spectinomycin), penicillin, streptomycin, ticarcillin, orany combination thereof. The antibiotics may be present in aconcentration of about 50 μg to about 800 μg per ml of semen, regardlessof whether the semen is neat, buffered, or contains additionalsubstances, such as for example, any of the additives mentioned herein.The Certified Semen Services (CS S) and National Association of AnimalBreeders (NAAB) have promulgated guidelines regarding the use ofantibiotics with respect to sperm collection and use.

A composition which regulates oxidation/reduction reactionsintracellularly and/or extracellularly may also be included in the spermsuspension. Such a composition may provide a protective effect to thesperm cells, such as for example by maintaining sperm viability orprogressive motility. Examples of such a composition include, forexample, pyruvate, vitamin K, lipoic acid, glutathione, flavins,quinones, superoxide dismutase (SOD), and SOD mimics. If included in thesperm suspension, such a composition may be present in a concentrationsufficient to effect the protective effect without detrimentallyaffecting sperm health. Exemplary concentration ranges include fromabout 10 μM to about 50 μM depending upon such factors as the particularcomposition being used or the concentration of sperm in the suspension.For example, pyruvate may be present in the sperm suspension in aconcentration from about 1 mM to about 50 mM, preferably from about 2.5mM to about 40 mM, more preferably from about 5 mM to 25 mM, even morepreferably from about 10 mM to 15 mM, still more preferably about 15 mM,and most preferably about 10 mM. Vitamin K may be present in the spermsuspension in a concentration from about 1 μM to about 100 μM,preferably from about 10 μM to about 100 μM, and more preferably about100 μM. Lipoic acid may be present in the sperm suspension in aconcentration from about 0.1 mM to about 1 mM, preferably from about 0.5mM to about 1 mM, and more preferably about 1 mM.

Staining of the Cells to be Sorted

Generally, sperm cells may be stained by forming a staining mixturecomprising sperm cells, a buffer, and a dye. The sperm cells may bederived from a freshly obtained semen sample, as discussed above withrespect to sample collection and dilution, or from a thawedcryopreserved semen sample.

If the semen sample is a thawed, previously cryopreserved sample, thesperm are preferably thawed immediately prior to staining. Generally, astraw or other cryopreservation vessel containing the frozen sperm maybe placed in a water bath, the temperature of which is preferably inexcess of the glass transition temperature of the sperm cell membrane(i.e., about 17° C.), but not so great as to adversely impact spermhealth. For example, frozen sperm may be thawed by immersing thecryopreservation vessel in a water bath maintained at a temperature ofabout 17° C. to about 40° C. for a period of about 30 seconds to about90 seconds.

Once obtained, the sperm cells may be introduced into the stainingmixture in the form of neat semen or in the form of a suspension derivedtherefrom, e.g., a sperm suspension as discussed above with respect tosample collection and dilution.

The dye may be in the form of a neat solid or a liquid composition. Thedye may also be dissolved or dispersed in an unbuffered liquid to form adye solution. Alternatively, the dye may be in the form of a dyesuspension comprising a dye and a buffer or buffered solution that isbiologically compatible with sperm cells. A range exemplary buffers andbuffered solutions are discussed above with respect to sample collectionand dilution. For example, among the buffers which may be used is a TCAbuffer solution comprising 3% TRIS base, 2% citric acid monohydrate, and1% fructose in water at a pH of about 7.0, or a carbonate-basedinhibitor solution comprising 0.204 g NaHCO₃, 0.433 g KHCO₃, and 0.473 gC₆H₈0₇.H₂O per 25 mL of purified water (0.097 moles/L of NaHCO₃, 0.173moles/L of KHC0₃, 0.090 moles/L C₆H₈0₇.H₂0 in water). Thus, for example,a staining mixture may be formed by combining neat semen with a dye.Alternatively, the staining mixture may be formed by combining neatsemen with a buffer or buffered solution and a dye. Additionally, thestaining mixture may be formed by combining a sperm suspension with adye.

The staining mixture may be formed by using one or more UV or visiblelight excitable, DNA selective dyes as previously described in U.S. Pat.No. 5,135,759 and WO 02/41906. Exemplary UV light excitable, selectivedyes include Hoechst 33342 and Hoechst 33258, each of which iscommercially available from Sigma-Aldrich (St. Louis, Mo.). Exemplaryvisible light excitable dyes include SYBR-14, commercially availablefrom Molecular Probes, Inc. (Eugene, Oreg.) and bisbenzimide-BODIPY®conjugate 6-{[3-((2Z)-2-{[1-(difluoroboryl)-3,5-dimethyl-1H-pyrrol-2-yl]methylene}-2H-pyrrol-5-yl) propanoyl] amino}-N-[3-(methyl{3-[({4-[6-(4-methylpiperazin-1-yl)-1H,3′H-2,5′-bibenzimidazol-2′-yl]phenoxy}acetyl)amino]propyl}amino) propyl] hexanamide (“BBC”) describedin WO 02/41906. Each of these dyes may be used alone or in combination;alternatively, other cell permeant UV and visible light excitable dyesmay be used, alone or in combination with the aforementioned dyes,provided the dye does not detrimentally affect the viability of thesperm cells to an unacceptable degree when used in concentrations whichenable sorting as described elsewhere.

The preferred concentration of the DNA selective dye in the stainingmixture is a function of a range of variables which include thepermeability of the cells to the selected dye, the temperature of thestaining mixture, the amount of time allowed for staining to occur, andthe degree of enrichment desired in the subsequent sorting step. Ingeneral, the dye concentration is preferably sufficient to achieve thedesired degree of staining in a reasonably short period of time withoutsubstantially detrimentally affecting sperm viability. For example, theconcentration of Hoechst 33342, Hoechst 33258, SYBR-14, or BBC in thestaining mixture will generally be between about 0.1 μM and about 1.0M,preferably from about 0.1 μM to about 700 μM, and more preferably fromabout 100 μM to about 200 μM. Accordingly, under one set of stainingconditions, the concentration of Hoechst 33342 is preferably about 100μM. Under another set of staining conditions, the concentration ofHoechst 33342 is about 150 μM. Under still another set of stainingconditions the concentration is preferably about 200 μM.

In addition to buffer, other additives may be included in the stainingmixture to enhance the viability or motility of the sperm; theseadditives may be provided as part of the sperm source, the dye source,or separately to the staining mixture. Such additives include energysources, antibiotics, compositions which regulate oxidation/reductionreactions intracellularly and/or extracellularly, and seminal plasma,the first three of which are discussed above with respect to samplecollection and dilution, and the last of which is discussed below withrespect to collection fluids. Such additives may be added during thestaining techniques in accordance therewith.

In particular, it has been observed that the inclusion of a compositionwhich regulates oxidation/reduction reactions intracellularly and/orextracellularly in the staining mixture may help to maintain spermviability at elevated staining temperatures, at elevated dyeconcentrations, at increased staining periods, or any combinationthereof. Examples of these compositions and the use of the same arediscussed above with respect to buffers and diluents. Such compositionsmay be added during the staining techniques in accordance therewith.

The staining mixture may be maintained at any of a range oftemperatures; typically, this will be within a range of about 4° C. toabout 50° C. For example, the staining mixture may be maintained at a“relatively low” temperature, i.e., a temperature of about 4° C. toabout 30° C.; in this embodiment, the temperature is preferably fromabout 20° C. to about 30° C., more preferably from about 25° C. to about30° C., and most preferable at about 28° C. Alternatively, the stainingmixture may be maintained within an “intermediate” temperature range,i.e., a temperature of about 30° C. to about 39° C.; in this embodiment,the temperature is preferably at about 34° C. to about 39° C., and morepreferably about 37° C. In addition, the staining mixture may bemaintained within a “relatively high” temperature range, i.e., atemperature of about 40° C. to about 50° C.; in this embodiment, thetemperature is preferably from about 40° C. to about 45° C., morepreferably from about 40° C. to about 43° C., and most preferably atabout 41° C. Selection of a preferred temperature generally depends upona range of variables, including for example, the permeability of thecells to the dye(s) being used, the concentration of the dye(s) in thestaining mixture, the amount of time the cells will be maintained in thestaining mixture, and the degree of enrichment desired in the sortingstep.

Uptake of dye by the sperm cells in the staining mixture is allowed tocontinue for a period of time sufficient to obtain the desired degree ofDNA staining. That period is typically a period sufficient for the dyeto bind to the DNA of the sperm cells such that X and Ychromosome-bearing sperm cells may be sorted based upon the differingand measurable fluorescence intensity between the two. Generally, thiswill be no more than about 160 minutes, preferably no more than about 90minutes, still more preferably no more than about 60 minutes, and mostpreferably from about 5 minutes to about 40 minutes.

Accordingly, in one embodiment, a staining mixture is formed comprisingsperm cells and a dye in a concentration from about 100 μM to about 200μM, and the staining mixture is held for a period of time at atemperature of about 41° C. In another embodiment, the staining mixturefurther comprises pyruvate in a concentration of about 10 mM, vitamin Kin a concentration of about 100 μM, or lipoic acid in a concentration ofabout 1 mM.

In still another embodiment, a staining mixture is formed comprisingsperm cells and a dye in a concentration from about 100 μM to about 200μM, and the staining mixture is held for a period of time at atemperature of about 28° C. In another embodiment, the staining mixturecomprises pyruvate in a concentration of about 10 mM, vitamin K in aconcentration of about 100 μM, or lipoic acid in a concentration ofabout 1 mM.

In yet another example, a staining mixture is formed comprising spermcells, a metabolic inhibitor comprising 0.204 g NaHCO₃, 0.433 g KHCO₃,and 0.473 g C₆H₈0₇-H₂0 per 25 mL of purified water (0.097 moles/L ofNaHCO₃, 0.173 moles/L of KHC0₃, 0.090 moles/L C₆H₈0₇H₂O in water), and adye in a concentration from about 100 μM to about 200 μM, and thestaining mixture is held for a period of time at a temperature of about28° C. In another embodiment, the staining mixture is held for a periodof time at a temperature of about 41° C.

Sheath Fluid

To sort the sperms cells, the stained cells are introduced as a samplefluid into the nozzle of a flow cytometer as described below. As part ofthe process, the sample fluid is typically surrounded by a sheath fluid.The sheath fluid permits the sperm cells in the sample fluid to be drawnout into a single file line as discussed below. The sheath fluid iscollected along with the sperm cells by the collection system of theflow cytometer and therefore forms part of the post-sort environment forthe sperm cells. Thus, it is desirable that the sheath fluid provides aprotective effect to the cells upon contact of cells by the sheathfluid.

The sheath fluid generally comprises a buffer or buffered solution.Examples of buffers and buffered solutions and illustrativeconcentrations of the same that may be used in the sheath fluid aredisclosed above with respect to sample collection and dilution. In aparticular embodiment, the sheath fluid comprises 0.96% Dulbecco'sphosphate buffered saline (w/v), 0.1% BSA (w/v), in water at a pH ofabout 7.0.

Optionally, the sheath fluid may also contain a range of additives thatare beneficial to sperm viability or motility. Such additives include,for example, an energy source, a protein source, an antibiotic, acomposition which regulates oxidation/reduction reactionsintracellularly and/or extracellularly, an alternative protein source,and polyvinyl alcohol. Each of these additives, and examples of thesame, is discussed above with respect to sample collection and dilution.Such additives may be added to the sheath fluid in accordance therewith.

The sheath fluid may optionally be filtered prior to the sorting step.Contaminants that may be present in the sheath fluid, such asnon-soluble particulates, may interfere with sorting. Therefore, thesheath fluid may be filtered prior to its introduction into a flowcytometer. Such filters and methods of using the same are well known inthe art. Generally, the filter is a membrane of about 0.1 microns toabout 0.5 microns, preferably about 0.2 microns to about 0.3 microns,and more preferably about 0.2 microns.

The stained cells may be introduced into the sheath fluid at any timesubsequent to staining. Typically, a stream of the stained cells in thesample fluid is injected into a stream of sheath fluid within the nozzleof the flow cytometer. Initially, there is substantially no contactingof the sample fluid and the sheath fluid due to laminar flow of thefluids as discussed in more detail below. It is desirable that thesample fluid and the sheath fluid remain as substantially discreteflowing streams until after the particles (e.g., the stained spermcells) in the sample fluid have been analyzed. At some point, however,the sheath fluid and the cells of the sample fluid come in contact withone another. For instance in a droplet sorting flow cytometer (discussedbelow) the sheath fluid and sample fluid begin contacting one another asdroplets are being formed downstream of the interrogation location.

At the time of the introduction of the stained cells and the sheathfluid, both the stained cells and the sheath fluid may be at atemperature from about 4° C. to about 50° C. The sheath fluid and thestained cells may be at the same or at different temperatures, witheither being at a higher temperature than the other. Accordingly, in oneembodiment, at the time of the introduction of the stained cells and thesheath fluid, both the cells and the sheath fluid are at the sametemperature; for example, at a “relatively low” temperature, such as forexample at about 5° C. to about 8° C.; at an “intermediate” temperature,such as for example at about 25° C. to about 30° C.; or at a “relativelyhigh” temperature, such as for example at about 40° C. to about 43° C.In another embodiment, the stained cells are at a higher temperaturethan the sheath fluid, such as for example, the cells being at about 40°C. to about 43° C. and the sheath fluid being at about room temperatureor at about 5° C. In yet another embodiment, the stained cells are at alower temperature than the sheath fluid.

Flow Cytometry

One embodiment of the present invention employs inventive technologiesin flow cytometry to analyze and sort the sperm cells. Referring now toFIGS. 2 and 3, one embodiment of a flow cytometry system of the presentinvention is designated in its entirety by the reference numeral 1. Aswill appear, the flow cytometry system 1 is useful for classifying andsorting particles, such as sperm cells, according to selectedcharacteristics. In general, the system 1 comprises a supply 3 ofcarrier fluid 17 containing particles to be sorted, a supply 7 of sheathfluid 19, flow cytometry apparatus having sorting capabilities,generally designated 9, and a fluid delivery system 15 for deliveringthe carrier 17 and sheath fluids 19 from respective supplies 3, 7 underpressure to the flow cytometry apparatus 9. The flow cytometry apparatus9 is adapted for receiving the carrier 17 and sheath 19 fluids, forcombining the fluids 17, 19 to create a stream of pressurized fluid 21,for directing the stream 21 carrying the particles through a focusedbeam of electromagnetic radiation 25 (e.g., UV laser light), and foranalyzing the electromagnetic radiation 31 (e.g., fluorescent light)emitted by particles passing through the focused beam 25. The apparatus9 also functions to break the stream 21 up into droplets 33 containingparticles to be evaluated, and to sort the droplets 33 based on theaforesaid measurements according to one or more characteristics of theparticles contained in the droplets 33. While this invention may be usedto analyze and preferably sort any type of particle, it has particularapplication to sorting cells according to one or more desiredcharacteristics of the cells (e.g., size, DNA content, shape, density,gene sequence, etc.). This invention is especially suited for sortinganimal sperm cells for commercial use by the animal production industryfor in vivo or in vitro artificial insemination, as discussed in moredetail below.

Single-Channel Sorting Apparatus and Method Flow Cytometry Apparatus

The flow cytometry apparatus 9 shown in FIG. 3 comprises a nozzlesystem, generally designated 101, for delivering a fluid stream 21containing particles (e.g., stained sperm cells) through a nozzleorifice 103 under pressure with the cells substantially in single fileand, in the case of sperm cells, with asymmetric heads of the spermcells substantially in a desired orientation which will be described. Asin conventional flow cytometry droplet sorting systems, a transducer 105is provided opposite the nozzle orifice 103 for introducing acousticalenergy into the fluid stream 21 which causes the stream 21 to break intodroplets 33 containing individual cells at a “droplet break-off”location 107 spaced from the nozzle orifice 103. The system 1 alsoincludes an optics system, generally designated 109, for focusing a beamof electromagnetic radiation 25 (e.g., 350-700 nm UV or visible laserlight) on the fluid stream 21 at an “interrogation” location 115 which,in the described embodiment, is between the nozzle orifice 103 and thedroplet break-off location 107. Thus, the described embodiment is ajet-in-air system. In other embodiments, the interrogation location 107could be inside the nozzle orifice 103 or upstream from the orifice 103.In any event, the cells are adapted to pass through the beam of light 25at the interrogation location 107, resulting in excitation of a chemicalstain (or other reporting medium) in the cells to cause fluorescenceemissions 31 having a wavelength different from that of the beam 25(e.g., if the illumination light 25 has a wavelength of about 350 to 370nm, the fluorescent emissions 31 may have a wavelength of about 460 nm).A photodetector 117 is operable to detect these emissions 31 and toconvert them into electrical signals which are processed and used toclassify the cells according to selected characteristics, such as theX/Y chromosome content of sperm cells. The flow cytometry apparatus 9further comprises a sorting system, generally designated 119, forsorting the droplets 33 into different groups or populations (e.g., twopopulations 123,125) according to the classification of the cellscontained in the droplets 33 and a collection system, generallydesignated 2201 (FIG. 2), for collecting the droplets 33 and maintainingthe segregation of the different populations 123,125.

Operation of the system 1 is controlled by a processor 131, such asmicroprocessor or other digital or analog control and/or processor, orcombinations thereof, which controls the various functions of thecomponents of the system 1 in a manner to be described. Significantly,the processor 131 is also responsive to particle analysis information tocontrol the output of the system 1 based on selected control and sortingstrategies involving different parameters, including the desired purityof one of the sorted populations of particles, the acceptable quantity(or percentage) of desired particles one of the populations as comparedto the quantity (or percentage) of desired particles in one or more ofthe other populations, and other parameters, as will be discussed later.

The various components of the system 1 are described in detail below.

Nozzle System

Referring to FIGS. 4 and 5, the nozzle system 101 comprises, in oneexemplary embodiment, a generally cylindrical flow body 133 having acentral longitudinal bore 135 through it, and a nozzle 137 on the flowbody 133 having a funnel-shaped nozzle body 139. A passage 141 extendsthrough the nozzle body 139 co-axial with the bore 135 in the flow body133 and terminates in the aforementioned nozzle orifice 103 at theforward end of the nozzle 137. The nozzle body 139 has an internallythreaded counterbore 145 at its rearward end for threadably receiving athreaded projection or stud 149 at the forward end of the flow body 133to removably connect the nozzle 137 to the flow body 133, the connectionbeing sealed by an O-ring seal 155. It will be understood that thenozzle can be removably connected to the flow body in other ways or,alternatively, the parts could be integrally formed as one piece.

Particles are delivered to the nozzle 137 by means of a conduit 157positioned co-axially in the bore 135 of the flow body 133. The outsidediameter of the conduit 157 is less than the inside diameter of the bore135 so that an annular space 167 is formed around the conduit 157. Inone particular embodiment, the conduit 157 is a tubular needle (e.g., a16-ga. needle having an inside diameter of 0.01 in.) having a front endwhich extends into the counterbore 145 at the back of the nozzle 137.The back end of the conduit 157 is connected to the fluid deliverysystem 15 for delivery of carrier fluid 17 (e.g., a staining mixturecontaining sperm cells) to the conduit 157. The annular space 167surrounding the conduit 157 is connected by means of a radial bore 173in the flow body 133 to the fluid delivery system 15 for delivery ofsheath fluid 19 into the annular space 167. As shown in FIGS. 3 and 5,an optional second radial bore 183 may be provided in the flow body 133connecting the annular space 167 to another line (not shown) for supplyof additional sheath fluid 19 to the nozzle 137.

As in conventional flow cytometry systems, sheath fluid 19 is introducedinto the annular space 167 surrounding the conduit 157. The velocity ofthe sheath fluid 19 as it flows past the tip of the conduit 157 is muchhigher that the velocity of the carrier fluid 17 exiting the conduit157, so that the carrier fluid 17 and cells (e.g., sperm cells)contained therein are accelerated by the sheath fluid 19 toward theorifice 103 of the nozzle 137. This acceleration functions to space thecells out generally in a single file arrangement for separate analysisby the optics system 109. The sheath fluid 19 surrounds the carrierfluid 17, resulting in the fluid stream 21 having a central core 189 ofcarrier fluid 17 and an outer co-axial sheath 191 of sheath fluid 19surrounding the central core 189 (see FIG. 6). As will be understood bythose skilled in flow cytometry, the laminar flow and hydrodynamicfocusing of the central core 189 tends to confine the particles to thecore 189, with little mixing of the sheath 19 and carrier fluids 17 inthe nozzle 137. Further, the central core 189 remains essentially intactwithin the sheath 191 as the stream 21 moves through the nozzle system101, until such time as droplets 33 are formed at the break-off location107. This type of co-axial flow is particularly suited for flowcytometry, because the particles to be analyzed are confined within therelatively narrow core 189 of the stream. As a result, a beam of light25 focused on the center or core 189 of the stream 21 will illuminatethe particles so that they may be analyzed substantially one at a time.By confining the core 189 within a sufficiently narrow diameter, one canobtain more uniform illumination of the particles in the core fluid 189.For good analytical results, the diameter of the core containing theparticles should desirably be within a range of 7 to 20 microns, andmore desirably within a range of 7 to 14 microns. The diameter of thecore stream 189 can be increased or decreased by adjusting the rate ofdelivery of the carrier fluid 17 relative to the rate of delivery of thesheath fluid 19.

Cell Orientation

For optimizing analytical results, it is desirable that particles havingasymmetric shapes be in a desired orientation when they pass through thelight beam from the optics system. As is known to those skilled in theart, fluorescence emissions from asymmetric particles tend toanisotropic (i.e., the intensity of the emissions are not uniform in alldirections). As used herein, the term “desired orientation” means anorientation which allows the processing system to discriminate betweencells having different characteristics with an accuracy in a range of70% to 100%, more desirably in a range of 80% to 100%, still moredesirably in a range of 90% to 100%, and most desirably 95% or greater.

To illustrate the point, a bovine sperm cell 201 is illustrated in FIGS.6 and 7. Typically, the cell has a paddle-shaped head 205 withrelatively flat wide opposite faces 207 and narrow edges 209, a nucleus213 in the head 205 containing the chromatic DNA mass of the cell, and atail 215 extending from the head 205 providing the motility necessaryfor effective fertilization. The average bovine sperm cell 201 has ahead length 219 of about 8 μm, a head width 221 of about 4 μm, and anoverall length 223 from the front of the head to the end of the tail ofabout 100 μm. In the average bovine sperm cell 201, the nucleus 213occupies most of the head volume and is only slightly smaller than thesperm head 205. Thus, the nucleus length 217 is almost equal to the headlength 219, again being about 8 μm in length. It has been observed thatin the bovine the X/Y chromosomes of the sperm cells 201 are localizedin a region of the nucleus 225 (FIG. 6) below and immediately adjacentthe longitudinal midline or equator 211 or center of the head 205. Morespecifically, this sub-equatorial region 225 extends no more than about20% of the nucleus length 217 on the lower half (toward the tail 215) ofthe nucleus 213, even more specifically no more than about 10-15% of thenucleus length 217 on the lower half of the nucleus 213, and still morespecifically no more than about 1.0-1.5 μm below the equator 211 of thenucleus 213.

When sperm cells pass through the excitation beam 25, it is desirablethat the cells be substantially in single file and that the head 205 ofthe each cell 201 be substantially similarly oriented to reduceorientation variability from cell to cell and thus provide for a moreuniform measurement of the cells. It is also desired that the cells havean orientation which will enable accurate discrimination between X and Ycells. Desirably, this orientation is one where the length of the spermcell 201 is generally aligned with the direction of stream flow 227(either head leading (shown FIG. 6) or head trailing) and where the head205 of the sperm cell 201 is rotated on its longitudinal axis so thatthe head 205 falls within an angular envelope 229 in which the lightbeam 25 from the optics system 109 will strike a wide face 207 of thecell 201 generally broadside, as shown schematically in FIG. 7, ratherthan a narrow edge 209 of the cell. Preferably, the envelope 229defining the desired orientation is generated by rotation of a spermcell 201 through an angular range of R1 relative to a plane P which isgenerally perpendicular to the incoming light beam 25, as viewed in across section taken transversely through the stream 21. The range R1 ispreferably 0 to 90 degrees, more preferably 0 to 60 degrees, and evenmore preferably 0 to 30 degrees. The nozzle of the present invention isconfigured to achieve this desired orientation with an accuracy of up to90% or more.

The tolerance for sperm orientation (i.e., the size of the envelope 229defined by angular range R1) is related to the numerical aperture of thelens used to collect fluorescence emissions 31 from the sperm cells. Inthe embodiment shown FIG. 7, for example, the optics system 109 has afluorescence emission 31 detection volume 579 defined by a solid angleof 55 degrees. When the rotational orientation of a sperm head 205 isoutside the envelope 229 defined by R1 as the sperm moves through thebeam 25, a relatively stronger fluorescence emission 31 from an edge 209of the sperm head 205 will be collected by the optic system 109,preventing the processor 131 from correlating the intensity of thefluorescence emission 31 with the X/Y chromosome content of the spermcell 201. However, the optics system 109 does not collect the relativelystronger fluorescence emissions 31 from the narrow edge 209 of the spermheads 205 as long as the rotational orientation of a sperm head 205 iswithin the envelope 229 as it passes through the interrogation location115. Thus, in the embodiment shown FIG. 7, the orientation of the spermcell does not result in collection of the relatively stronger edge-wisefluorescence emissions as long as the narrow edges 209 of the sperm head205 are confined within angle R1. The solid angle of the collectionvolume 579 can be decreased by using a lens with a smaller numericalaperture, thereby increasing angle R1 and the tolerance for poorlyoriented sperm. However, this also decreases the number of photons thatcan be collected by the optics system 109, which can impact themeasurement of fluorescence emissions 31 by reducing the intensity ofthe emissions 31 detected by the photodetector. Likewise, if the opticssystem 109 collects fluorescence emissions 31 with a high numericalaperture lens to obtain a stronger intensity of the fluorescenceemissions detected by the photodetector, then the tolerance for spermorientation decreases. Thus, in designing a system of the presentinvention, one needs to strike a balance between the tolerance for spermorientation and the numerical lens aperture. The optimal balance willdepend on the orienting capabilities and optical sensitivity of thesystem. In one desirable embodiment, for example, a lens having anumerical aperture 0.65 is used.

Nozzle Design

In one embodiment, as shown in FIGS. 8 and 9, the interior 231 of thenozzle body 139 downstream from the counterbore 145 has an interiorsurface 233 comprising first, second and third axially tapered regions235, 237, 239 for progressively accelerating the speed of the fluidstream 21 in a downstream direction toward the nozzle orifice 103. Asnoted previously, this acceleration functions to space the particles(e.g., cells) in the stream 21 so they assume a generally single fileformation so they can be analyzed substantially one particle at a time.At least two of these regions, and preferably all three 235, 237, 239,have generally elliptical (oval) shapes in cross sections taken at rightangles to the longitudinal axis 247 of the nozzle 137, as is shown inFIGS. 9A-9H and FIGS. 9J-9K. The interior surface 233 of the nozzle body139 also has a fourth region 249, not tapered, downstream from the firstthree regions 235, 237, 239 and immediately upstream of the nozzleorifice 103 which, in one embodiment, is formed in a separate orificemember 255 secured in a counterbore 257 at the front of the nozzle body139. In one embodiment, the generally elliptical cross sectional shapesof the first 235 and second 237 regions are oriented in substantiallythe same direction to define a first torsional zone 259, and thegenerally elliptical cross sectional shape of the third region 239,constituting a second torsional zone 261, is oriented at an angle (e.g.,about 90 degrees) relative to the generally elliptical cross sectionalshapes of the first 235 and second 237 regions. The orientation is suchthat the interior surface 233 of the nozzle body 139 applies torsionalforces to the fluid stream 21 and thereby tends to orient the spermcells 201 in the aforestated desired orientation as they pass throughthe nozzle orifice 103. Preferably, the first torsional zone 259 has anaxial length 273 of 3.0-4.5 mm, preferably about 3.6 mm, and the first235 and second 237 tapered regions making up the zone 259 haveapproximately equal axial lengths 275, 277 (e.g., about 1.8 mm). Thesecond torsional zone 261 has an axial length 279 of 3.5-5.0 mm,preferably about 4.45 mm. The fourth region 249 is preferably generallycylindrical in shape. Each generally cross-sectional elliptical shapeA-D (FIG. 8) at the boundaries of the first 235, second 237 and third239 regions has a major axis diameter and a minor axis diameter,exemplary dimensions of which are shown in FIG. 8 and Table II below.

TABLE II Major Axis Minor Axis Diameter Diameter Ellipse (mm) (mm) RatioA 7.0 6.0 1.2 B 6.1 5.3 1.15 C 2.1 2.1 1 D 0.9 0.2 1.45

It will be understood that the above dimensions are exemplary, and thatother dimensions and shapes may also be suitable. Functionally, thechanges in the ratios between the major and minor diameters, and thedifferent orientations of the elliptical shapes of the regions, createside forces which act on each cell 201 and apply a torsional force 271tending to rotate the cell 201 on its longitudinal axis so that its widefaces 207 align with the minor axis in the first torsional zone 259 andas the cell is gently twisted (e.g., 90 degrees) to align with the minoraxis of the second torsional zone 261. Each of the tapered surfaces 235,237, 239 also serves to accelerate the stream 21 (and cells) flowingthrough the nozzle 101. In one embodiment, the acceleration increasesmore gradually in the first 235 and third 239 regions and more rapidlyin the second region 237. Again by way of example, the taper of thefirst region 235 may range from about 11-14 degrees; the taper in thesecond region 237 may range from about 42-48 degrees; and the taper inthe third region 239 may vary from about 8-12 degrees. The nozzle body139 is formed from a suitable material such as molded plastic (ABS) ormetal.

The orifice member 255 (FIG. 8) is preferably formed from a hard, wearresistant material, such as sapphire, which is capable of being machinedor otherwise formed with precise dimensions. The orifice member 255itself has, in one embodiment, a conical upstream surface 309 ofgenerally circular cross section which decreases in diameter from about0.92 mm to about 0.060 mm and has an axial length 317 of about 0.54 mmand a taper angle of about 39 degrees. The orifice member 255 also has agenerally cylindrical downstream surface 315 with a diameter of about0.060 mm and an axial length 327 of about 0.36 mm. These dimensions areexemplary only, and it will be understood that the orifice member 255may have other sizes and shapes. For example, the shape of the upstreamsurface 309 may be generally elliptical (oval) in cross section, and thediameter of the orifice 103 at the downstream end of the nozzle 137 mayrange from 40 to 100 microns or more. It is desirable that the size ofthe orifice 103 be such that the cells exiting the nozzle 101 aresubstantially in single file formation within the core 189 of the stream21 and substantially in the desired orientation, as describedpreviously. For example, in the case of sperm cells an orifice 103having a diameter of about 60-62 microns at the downstream end has beenfound to be suitable. Preferably, the nozzle orifice 103 serves tofurther accelerate the stream 21 and to shape and size the stream 21 foroptimum cell spacing, cell orientation and droplet 33 formation, as willbe described.

The velocity of the cells as they exit the nozzle 137 will depend onvarious factors, including the pressure at which sheath fluid 19 isintroduced into the nozzle system 101. At a pressure of 20 psi, thecells will exit the nozzle orifice 103 of the above embodiment at avelocity of about 16.6 m/s as a generally cylindrical stream 21containing cells which are substantially similarly oriented at the core189 of the stream 21. At a sheath pressure of 30 psi, the cell velocitywill be about 20.3 m/s. At different sheath fluid 19 pressures, thevelocity of the stream 21 will vary.

Introduction of Core Stream to Torsional Zone

Improved orientation of particles may be obtained by altering the flowof the fluid stream 21 through an orienting nozzle so that the corestream 189 containing the particles to be oriented (e.g., sperm cells)is directed along a flow path, at least a portion of which is offsetfrom the center of the nozzle so that the particles are subjected to thehydrodynamic orienting forces generated by a nozzle while they are at alocation that is offset from the center of the nozzle. Directing thecore stream 189 along an offset flow path may also improve orientationof particles in a traditional nozzle (i.e., one that does not have anytorsional zones). In many nozzles, one can determine that a givenposition is offset from the center of the nozzle because it is displacedfrom a longitudinal axis of the nozzle. One can also recognize that aparticular position is offset from the center of a nozzle because it isdisplaced from the geometric center of a cross sectional area of thenozzle through which the fluid stream flows.

A number of techniques may be used to direct the core stream 189 along aflow path that is offset from the center of the nozzle. For example, anorienting baffle may be positioned in the nozzle to deflect the corestream to one side of the nozzle. Similarly, the conduit 157 forintroducing the core stream 189 containing the sample particles may berelocated from the traditional center of the nozzle to an offsetlocation. Furthermore, it is contemplated that an offset sampleintroduction conduit 157 may be used in combination with an orientingbaffle. Exemplary embodiments of use of an orienting baffle and use ofan offset sample introduction conduit are discussed below.

The improved orientation of particles (e.g., sperm cells) achieved byuse of an orienting baffle and/or offset sample introduction conduit 157may be due to a number of factors. One factor is that the deflection ofthe core stream 189 and/or a change in the size and shape of the crosssectional flow area results in application of hydrodynamic forces thattend to orient asymmetric particles. (Kachel, et al., Histochemistry andCytochemistry, 25 (7): 774-80 (1977)). Another factor is that it hasbeen found that asymmetric particles (in particular sperm cells) tend toorient as they flow in a fluid stream in close proximity to a solidsurface. Thus, by directing the core stream 189 so that it is in closeproximity to the interior surface of a nozzle or a baffle surface onecan obtain improved orientation of the particles. Furthermore, a baffleand/or offset sample introduction conduit can be used in conjunctionwith an orienting nozzle which applies additional orienting forces(e.g., torsional forces) to the asymmetric particles. In that case, thebaffle can operate to direct the fluid stream so that the core streamcontaining the particles to be oriented flows along a path that isoffset from the center of the nozzle while the particles are subjectedto the torsional forces generated by one or more of the torsional zones.

Orienting Baffle

FIGS. 10-13 show one exemplary orienting baffle, generally designated2001, positioned in the orienting nozzle 137 described above. However,the baffle 2001 could be used in conjunction with a different nozzle,including a non-orienting nozzle, without departing from the scope ofthis invention. The baffle 2001 is positioned in the nozzle upstreamfrom the orifice 103 and downstream from the sample injection needle157. Referring to FIGS. 14 and 15, the baffle comprises a baffle plate2003 that is held in place by a baffle holder 2005. In the embodimentshown, the baffle plate 2003 is generally L-shaped and constructed of asubstantially rigid, durable and corrosion-resistant material (e.g.,stainless steel). The L-shaped plate 2003 has an upstream leg 2007 and adownstream leg 2009, which are desirably substantially perpendicular toeach other (e.g., within about 5 degrees of being perpendicular). In theexemplary embodiment shown in the drawings, the two legs 2007, 2009 ofthe L-shaped plate 2003 intersect at a line 2015 that is perpendicularto the longitudinal axis 2017 of the nozzle 137 (FIG. 11). As shown inFIG. 14, the line of intersection 2015 is also spaced a short distance2033 (e.g., about 0.3 mm) away from the longitudinal axis 2057 of thebaffle holder 2005. The upstream leg 2007 of the L-shaped plate 2003extends from the line of intersection 2015 away from the longitudinalaxis 2017 of the nozzle 137 all the way to the edge of the baffle holder2005, as shown in FIG. 15. Thus, the upstream leg 2007 is formed with acurved edge 2019 that closely matches the shape of the baffle holder2005. As shown in FIG. 14, the upstream leg 2007 is inclined at an angleAA of about 15-25 degrees from perpendicular to the longitudinal axis2057 of the baffle holder 2005. The downstream leg 2009 of the L-shapedplate 2007 extends downstream from the line of intersection 2015 of thetwo legs 2007, 2009 a distance 2025 of about 2.0-2.5 mm at an angle BBthat is in the range of about 60-80 degrees from perpendicular to thelongitudinal axis 2057 of the baffle holder 2005.

The baffle holder 2005 is sized and shaped to fit inside the nozzle 137,as shown in FIGS. 10-13. The baffle holder 2005 is preferably made of amoldable material (e.g., polypropylene) although the baffle holder 2005may be constructed from other materials without departing from the scopeof the present invention. The baffle holder 2005 used in the exemplaryembodiment, shown in FIGS. 14 and 15, is generally shaped as a hollowcylindrical shell about 4.0-4.5 mm in overall length 2027. The baffleholder 2005 has an exterior diameter 2029 of about 5-6 mm and aninterior diameter 2031 of about 2.5-3.5 mm. If the baffle holder 2005 isto be molded, a minor draft (not shown) can be provided on the surfacesof the holder 2005 (e.g., to allow the baffle holder to be easilyremoved from an injection molding machine). The upstream end 2035 of theexemplary baffle holder 2005 has an inclined surface 2037 which isinclined at the same angle AA as the upstream leg 2007 of the L-shapedplate 2003. The upstream leg 2007 of the L-shaped plate 2003 abutsagainst and is supported by the inclined surface 2037 of the baffleholder 2005. The side edges 2039 (FIG. 15) of the downstream leg 2009 ofthe L-shaped plate 2003 are partially embedded (e.g., received in slots)in the baffle holder 2005 to hold the baffle plate 2003 in a position inwhich the downstream leg 2009 spans generally from one side of thebaffle holder 2005 to the other. The downstream edge 2041 of thedownstream leg 2009 in the exemplary embodiment forms a straight linewhich is generally perpendicular to the longitudinal axis 2057 of thebaffle holder 2057. There is a gap 2049 (FIG. 14) between the downstreamedge 2041 of the downstream leg 2009 and the interior cylindricalsurface 2051 of the baffle holder 2005. The gap 2049 provides fluidcommunication between a volume 2053 defined by the legs 2007, 2009 ofthe L-shaped plate 2003 and the interior cylindrical surface 2051 of thebaffle holder 2003 and the rest of the interior volume 2055 of thenozzle 137.

The baffle holder 2005 is desirably positioned inside the nozzle withthe longitudinal axis 2057 of the baffle holder 2005 generally alignedwith the longitudinal axis 2017 of the nozzle 137 so that it holds theL-shaped plate 2003 in the position described above. Desirably, theexemplary baffle plate 2003 is rotationally oriented so that the line ofintersection 2015 of the two legs 2007, 2009 of the plate 2003 isparallel to a line 2059 running through the major axis of ellipse D, asshown in FIG. 16. However, the exemplary baffle 2001 also performs wellwhen the intersection 2015 of the two legs 2007, 2009 of the L-shapedplate 2003 is perpendicular to the line 2059 running through the majoraxis of ellipse D, as shown in FIG. 17. Furthermore, the baffle may haveany rotational orientation without departing from the scope of thisinvention. As shown in FIG. 12, the sample injection needle 157 in theexemplary embodiment is desirably a distance 2061 of about 0.25-1.0 mmupstream from the most upstream portion 2035 of the baffle 2001. Moredesirably, the sample injection needle 157 is about 0.55-0.65 mmupstream from the most upstream portion 2035 of the baffle 2001.

The baffle holder 2005 may be held in a desired position relative to thenozzle in any number of ways. Referring to FIG. 14, the downstream end2067 of the baffle holder 2005 is stepped so that it fits fartherdownstream in the nozzle 137. The stepped downstream end 2067 of theholder 2005 is circular in shape and abuts against the ellipticallyshaped interior surface 233 of the nozzle 137. Thus, the contact betweenthe interior surface 233 of the nozzle 137 and the baffle holder 2005 isgenerally limited to two points 2069, as shown in FIG. 13. A pair ofO-rings 2071 are positioned around the baffle holder 2005 between thenozzle 137 and the threaded projection 149 of the flow body 133 (FIGS.11-13) and seal the nozzle system 101 against leakage. The O-rings 2071may be made of Viton®, or any other similar materials. The two O-rings2071 are compressed as the nozzle 137 is screwed onto the threadedprojection 149 to provide a fluid-tight seal. Two O-rings 2071 are usedin the exemplary embodiment because a single O-ring cannot be compressedwithin the space between the nozzle 137 and the flow body 133 due to thelength 2027 of the baffle holder 2005. Any number of O-rings or adifferent type of seal could be used without departing from the scope ofthe present invention, provided that the number of O-rings or other typeof seal is selected so that there will be a fluid-tight seal when thenozzle 137 is screwed onto the flow body 133. This will depend on anumber of factors, including the size and shape of the nozzle 137, flowbody 133, baffle holder 2005, and O-rings 2071 as well as the type ofseal. The O-rings 2071 also help hold the baffle holder 2005 in thedesired position. The O-rings 2071 occupy the space around the baffleholder 2005, thereby restricting side-to-side movement of the baffleholder 2005 inside the nozzle 137. Frictional forces between the O-rings2071 and the baffle holder 2005 also resist rotational movement of thebaffle holder 2005.

When the nozzle 137 is tightened on the flow body 133 as shown in FIG.12, the downstream end 2077 of the threaded projection 149 from the flowbody 133, in the form of a boss in this embodiment, is approximatelyeven with the most upstream portion 2035 of the baffle 2001. As aresult, the baffle holder 2005 is held axially captive between the flowbody 133 (at the upstream end 2035 of the baffle holder 2005) and theinterior surface 233 of the nozzle 137 (at the downstream end 2067 ofthe baffle holder 2005). Other retaining mechanisms may be used. In theembodiment shown in the drawings, the interior diameter of the boss 2079(FIG. 12) at the downstream end of the threaded projection 149 isroughly equal to the internal diameter 2031 of the baffle holder 2005.

Those skilled in the art will recognize that the flow through the nozzlesystem 101 remains laminar notwithstanding the baffle 2001 because thesmall cross sectional area through which the fluids must flow results ina low Reynolds number for the flow. As is shown in FIG. 11, the baffledeflects the core stream 189 and sheath stream 191 away from the centrallongitudinal axis 2017 of the nozzle 137 and toward an interior surface233 of the nozzle 137. In one embodiment, the core stream 189 also flowsvery close to the interior surface 233 of the nozzle 137 as the corestream 189 passes between the transition between the first 259 andsecond 261 torsional zones. However, a portion 2081 of the sheath fluidstream 191 remains between the core stream 189 and the interior surface233 of the nozzle 137 so the particles in the core stream 189 do notactually impact or contact the interior surface 233 of the nozzle 137.Farther downstream in the nozzle 137, the hydrodynamic forces push thecore stream 189 back toward the center of the nozzle 137 (e.g., inalignment with the longitudinal axis 2017 of the nozzle 137).

Referring to FIGS. 18A-18E, the baffle 2001 changes the shape andreduces the size of the cross sectional flow area in the nozzle 137.(For the sake of clarity, FIGS. 18A-18E do not show any nozzle structuredownstream from the baffle. The flow area in each of the FIGS. 18A-18Eis outlined in bold for clarity.) Upstream from the baffle 2001 (FIG.18A), the cross sectional flow area 2087 is generally circular orelliptical. At the upstream end 2035 of the baffle 2001, the flow areabegins to change from a circular shape to a generally semi-circularshape 2089 at the intersection 2015 of the legs 2007, 2009 of the baffleplate 2003 (FIG. 18B), although other shapes may be suitable. There thecross sectional flow area 2089 is smaller than the flow area 2087upstream from the baffle. FIG. 18C illustrates the flow area 2091 asfluid flows through a part of the baffle holder 2005, and FIG. 18Dillustrates the flow area 2093 farther downstream at the downstream end2041 of the downstream leg 2009 of the baffle plate 2003. It will beobserved that flow area 2093 is somewhat larger than flow area 2091 dueto the angular orientation of the downstream leg 2009 of the baffleplate 2003. Downstream from the baffle plate 2003 (FIG. 18E) the flowarea 2094 through the baffle corresponds the shape of the interiorsurface 2051 of the baffle holder 2005, which is circular in theillustrated embodiment. (Other shapes may be suitable.) Downstream fromthe baffle holder 2005 the torsional zones 259,261 of the nozzle 137desirably provide torsional forces as discussed above.

As shown in FIG. 11, it has been observed that one or more air bubbles2095 may become trapped in the volume 2053 between the downstream leg2009 of the L-shaped plate 2003 and the baffle holder 2005. Furthermore,a portion of a bubble 2095 may extend through the gap 2049 between theedge 2041 of the downstream leg 2009 and the baffle holder 2005. Thus,the air bubble(s) 2095 can occupy a portion of the cross sectional flowarea downstream of the downstream leg 2009 of the L-shaped plate 2003,perhaps affecting the flow of fluid through the nozzle 137. Theexemplary baffle 2001 has been found to work well both with and withoutthe air bubble(s) 2095. Thus, a baffle can be used to orient sperm cellswithout involvement of any bubbles without departing from the scope ofthe present invention.

Another exemplary orienting baffle, generally designated 2097, is shownin FIGS. 19 and 20. The baffle 2097 comprises a flat generallysemi-circular baffle plate 2099 in the orienting nozzle 137 discussedabove. The baffle plate 2099 is positioned in the nozzle 137 downstreamof the sample introduction conduit 157 and generally perpendicular tothe longitudinal axis 2017 of the nozzle 137. The baffle plate 2099 hasa curved edge 2101 that generally matches the curvature of the interiorsurface 233 of the nozzle 137 so that there are no large gaps betweenthe curved edge 2101 of the baffle plate 2099 and the interior surface233 of the nozzle 137. The baffle plate 2099 also has a straight edge2103 that extends a short distance past longitudinal axis 2017 of thenozzle 137 so that it is approximately aligned with the outer diameter2109 of the sample introduction conduit 157. The baffle plate 2099 isheld in position by friction resulting from compression of the baffleplate 2099 between an O-ring seal 2105, which is similar to the o-ringseals 2071 described in connection with the L-shaped baffle 2001 above,and an annular shoulder or shelf 2107 formed on the interior of thenozzle 137. As shown in FIG. 19 the orienting baffle 2099 operates bydeflecting the fluid stream so that the core stream 189 containing theparticles to be analyzed is offset from the central longitudinal axis2017 of the nozzle 137 along a portion of its flow path. For example,the core stream 189 may be directed along a flow path that is offsetfrom the longitudinal axis 2017 of the nozzle 137 as it flows throughthe first torsional zone 259, as well as at least a portion of thesecond torsional zone 261. Consequently, the particles (e.g., spermcells) are subjected to the torsional forces generated by the torsionalzones 259, 261 while they are in a position that is offset from thecentral longitudinal axis 2017 of the nozzle 137.

Those skilled in the art will recognize that substantial changes may bemade to the exemplary baffles 2001, 2097 described above withoutdeparting from the scope of the present invention. All that is requiredis that the baffle be configured to deflect the core stream 189 andsheath stream 191 toward an interior surface of the nozzle or to causethe core 189 and sheath stream 191 to flow through a cross sectionalarea that changes in size and/or shape. Further, it is understood thatthe orienting baffle structure may be integrally formed with the nozzleor integrally formed with the nozzle and flow body without departingfrom the scope of the present invention.

Offset Sample Introduction Conduit

The core stream 189 may be directed along a flow path that is offsetfrom the central longitudinal axis 2017 of the nozzle 137 byrepositioning the sample introduction conduit 157 from its traditionalposition at the center of the nozzle 137 to an offset position. Forexample, FIG. 21 shows an exemplary offset sample introducing nozzlesystem 2151 having an offset sample introduction conduit 157. Except asnoted, the nozzle system 2151 is substantially the same as the nozzlesystem 101 shown in FIGS. 4 and 5. The significant difference is thatthe sample introduction conduit 157 has been moved away from the centerof the nozzle 137 so that it is no longer aligned with the nozzle'slongitudinal axis 2017. Thus, the core stream 189 is directed into thetorsional zones 259, 261 of the orienting nozzle 137 along a flow paththat is offset from the longitudinal axis 2017. Although the exemplarynozzle system 2151 shown in FIG. 21 uses the exemplary orienting nozzle137 describe above, it is contemplated that offset sample introductionconduit 157 could be used with a different orienting nozzle or anon-orienting nozzle to orient particles in the core stream 189.

Nozzle Mounting and Adjustment

The flow body 133 and nozzle 137 are mounted in a selected orientationand position by means of a nozzle mount, generally designated 331. Inone embodiment (FIG. 22), the mount 331 comprises a plurality of stages,including first and second linear stages 333, 337 providing linearadjustment of the flow body 133 and nozzle 137 along X and Y axes 339,341, respectively, and a third rotational stage 343 providing rotationaladjustment about a Z axis 345 corresponding to the longitudinal axis2017 of the flow body 133 and nozzle 137. These stages 333, 337, 343 maybe conventional in design, suitable stages being commercially available,for example, from Newport Corporation of Irvine Calif. In particular,the first linear motion stage 333 comprises a fixed first stage member(not shown) mounted on a frame 349, a movable first stage member 355slidable on the fixed first stage member along the X axis 339, and anactuator 357, e.g., a micrometer, for precisely moving the movable firststage 355 member to a selected X-axis position. The second linear motionstage 337 comprises a fixed second stage member 359 mounted on themovable first stage member 355, a movable second stage member 361slidable on the fixed second stage member 359 along the Y axis 341, andan actuator 363, e.g., a micrometer, for precisely moving the movablesecond stage member 361 to a selected Y-axis position. The rotational(third) stage 343 comprises a fixed third stage member 365 mounted onthe movable second stage member 316, a movable third stage member 371rotatably mounted on the fixed third stage member 365 for rotation aboutthe Z-axis 345, and an actuator 373, e.g., a micrometer, for preciselyrotating the movable third stage member 371 to a selected angularposition relative to the Z-axis 345. The three-axis adjustment providedby these stages 333, 337343 allows the nozzle 137 and the fluid stream21 exiting the nozzle orifice 103 to be precisely positioned relative tothe optics system 109. Rotation of the nozzle 137 about the Z-axis 345is particularly helpful because it enables the stream 21 exiting thenozzle 137 to be rotated to bring the cells (e.g., sperm cells) orientedby the nozzle 137 into a position in which the light beam 25 from theoptics system 109 will fall on the desired surfaces of the cells (e.g.,the flat faces 207 of sperm heads 205), as illustrated schematically inFIG. 23. Other nozzle mounts may be suitable. For example, a 4-axisnozzle mounting system can also be used, providing linear adjustmentalong X, Y and Z axes and rotational adjustment along the Z axis.Further, it may be desirable to use one or more stages having anautomated alignment feature, such as a servo or stepper motor controlledmicrotranslation stage (e.g., part number M-110.2DG from Polytech PI,Inc. of Auburn, Mich.).

In one embodiment shown schematically in FIG. 36, for example, thenozzle 137 is oriented to direct a stream 21 containing cells to beanalyzed in a generally upward direction. The angle 377 between thedirection of the fluid stream 21 and horizontal is preferably in therange of 5 to 85 degrees, more preferably in the range of 15 to 75degrees, even more preferably about 30 to 65 degrees, still morepreferably about 45 to 60 degrees, and most preferably about 50 to 55degrees. This orientation is advantageous in that any air trapped in thenozzle system 101 is readily removed. Also, the velocity of the fluidstream 21 decreases gradually under the force of gravity prior tocollection of the droplets 33. A more gradual deceleration of thedroplets 33 is believed to be less stressful to the cells being analyzedwhich, in the case of sperm cells, can result in higher motility of thesorted sperm after collection. Of course, in other embodiments of thepresent invention, the nozzle 101 is positioned so that the fluid stream21 has a substantially downward velocity when it exits the orifice 103as is conventional for jet-in-air cytometers.

Optionally, components of the nozzle system 101 such as the flow body133 and nozzle 137 are coated with a non-reflective, non-emissivematerial (e.g., a dull dark paint or epoxy which does not emit lightwhen subjected to UV laser light) to reduce any reflected and/or emittedlight off these elements 133,137 which might otherwise cause signalnoise or have other adverse effects on the optics system 109.

Transducer and Droplet Formation

The transducer 105 for introducing energy into the fluid stream 21comprises, in one embodiment, a collar 379 containing a piezoelectricelement (not shown) secured around the flow body 133 of the nozzlesystem 101 (FIGS. 3-5). The transducer is of conventional design, suchas is available from Beckman Coulter, Inc. as part No. 6858368. Thetransducer has terminals 383 for connection to a suitable source ofacoustical energy so that energy can be delivered to the fluid stream 21at a frequency which will cause it to break into droplets 33 at thedroplet break-off location 107 downstream from the nozzle 137 a distanced (FIG. 24). As will be understood by those skilled in flow cytometry,the characteristics of the droplet formation are governed by thefollowing Equation 1:

(V=fλ)  Equation 1

Where V is the velocity of the stream 21; f is the frequency applied tothe fluid stream 21 through the nozzle 137; and λ is the “wave length”or distance between the droplets 33. It is a known principle of flowcytometry that droplets 33 will form in a regular pattern with thedistance between droplets 33 being 4.54 times the diameter of the stream21. Since the diameter D of the stream 21 close to the nozzle 137generally corresponds to the diameter of the nozzle orifice 103 at itsdownstream end, the frequency at which the stream 21 (and nozzle 137)must be vibrated to form the droplets 33 can be easily calculated usingthe following Equation 2:

(f=V/4.54D)  Equation 2

The transducer 105 may be operated to generate in the range of30,000-100,000 droplets 33 per second. For example, the transducer 105may generate 50,000-55,000 droplets per second. Assuming the frequencyis 55,000 cycles per second (55 kHz), and further assuming that theconcentration of cells in the stream 21 is such that cells exit thenozzle 137 at a substantially matching rate of 55,000 cells per second,then there will be, on average, one cell per droplet 33. (In reality,some droplets 33 will contain no cells, some will contain one cell, andsome will contain more than one cell.) Of course, any of various factorscan be changed to vary this average, including a change in frequency(f), stream 21 (orifice 103) size (D) and stream 21 velocity (V).Ideally, these factors should be such as to reduce the amount of stressimparted to the cells during the course of the process, especially inthe case of sperm cells where the preservation of motility is important.

Break-Off Sensor

Referring to FIG. 2, a break-off sensor 389 may be employed to determinethe location (e.g., break-off location 107) at which the stream 21begins to form free droplets 33. The break-off location 107 will varydepending on several factors including stream 21 viscosity, surfacetension of the fluid and the amplitude of vibration of the transducer105. By monitoring the break-off location 107, the amplitude of thetransducer 105 may be varied to maintain the break-off location 107within a given range so that the time at which each droplet 33 breaksoff can be more accurately predicted by the microprocessor 131. Thisallows the microprocessor 131 to accurately control the electricalcharge of the droplet 33 which is accomplished by selectivelycontrolling the charge of the stream 21. Since the charge of the droplet33 will be the same as the charge of the stream 21 immediately beforedroplet 33 formation, the microprocessor 131 controls the sorting of thedroplets 33 by selectively charging the stream 21, as noted below.

In general, a break-off sensor is for use with any continuous stream offluid which is breaking into droplets at a break-off location. (In theembodiment of FIG. 2, the break-off sensor 389 is located downstreamfrom the nozzle 137 and interrogation location 115.) One exemplarybreak-off sensor 389 is shown schematically in FIG. 25. A light source393 is positioned on one side of the stream 21 to illuminate the stream21 within the given range at which the break-off location 107 will bemaintained. A linear photoarray 395 positioned on the other side of thestream 21 is adapted to be oriented along an axis substantially parallelto the stream 21. As a result, the photoarray 395 detects light from thelight source 393 which passes through the droplets 33 and providesoutput signals corresponding to the detected light.

The output signals are processed to determine the position of thebreak-off location 107. For example, the output signals may be digitizedand provided to the processor 131 for processing. Alternatively, asshown in FIG. 25, the light source 393 may be an LED or other sourcewhich generates a near-infrared portion of the visible spectrum. Thelight passing between the droplets 33 is magnified by a lens 401 anddirected toward an 8 by 1 linear array of photodiodes 395. Eachphotodiode generates a current that is proportional to the lightintensity impinging thereon. This current is fed into 8 current tovoltage op-amp circuits 405. The output voltage from the op-amps is ACcoupled into 8 track/hold amplifiers 407. The track/hold signal 409 usedby the amplifiers is taken from the transducer 105. The output from thetrack/hold amplifier is fed into the A/D converter 411 of amicroprocessor unit (MPU) 391. The digital values computed by the MPU391 will be provided to the system control microprocessor 131. A lookuptable and/or algorithm may be used by the system control microprocessor131 to convert between break-off location 107 drift and voltageadjustment to the transducer 105. Alternatively, the output from the MPU391 may be an analog signal such as a DC voltage having an amplitudecorresponding to a change in the amplitude of vibration of thetransducer 105. The dc voltage can be applied to the high voltageamplifier input driving the droplet transducer 105 to vary the amplitudeof vibration. Thus, such a processor 391 would constitute a control forreceiving the output signal from the photoarray 395 and providing alocation signal corresponding to a location of the break-off location107. Such a processor 391 would also constitute a control for receivingthe output signal indicative of the position of the break-off location107 of the droplets 33 and varying operation of the transducer 105 as afunction of the position of the location 107.

Alternatively, as is well known to those skilled in the art, a videocamera and strobe light may be used to monitor and control the dropletbreak-off location. Thus, as shown in FIGS. 26-27, a video camera system412 and strobe 413 may be provided to monitor the break-off location107. It is desirable to place the strobe 413 behind a mask 414A (e.g., acover with a small slit; shaped opening 414B) to limit the amount oflight produced by the strobe 413 that enters the optics system 109 (FIG.27).

Epi-Illumination Optics System

The optics system 109 is adapted for focusing a beam of electromagneticradiation 25 (e.g., a laser beam) on the fluid stream 21 as a beam spot,so that the cells to be analyzed pass through the spot. The beam 25 maybe laser light in the visible or ultraviolet portion of the spectrum,for example, having a wavelength of about 350-700 nm, although otherwavelengths may be used. The wavelength of the laser light may beselected so that it is capable of exciting a particular fluorochromeused to analyze particles. If the optics system 109 is used to analyzesperm cells stained with Hoechst 33342, for instance, the wavelength maybe selected to be in the range of about 350-370 nm. The power output ofthe laser may vary between 50 and 300 mW. Sperm cells may be analyzedusing a 200 mW laser, for example. Referring to FIGS. 28-34, the system109 is an epi-illumination system 415 comprising an instrument,generally designated 417, having a longitudinal optical axis 419. Asused herein, the term “epi-illumination” means an optics system where atleast some of the fluorescence emissions from cells passing through thebeam spot are directed back through the optical instrument along thesame axis as the focused beam 25, but in the opposite direction. Thistype of system is advantageous in that only one set of optics isrequired, including only one photodetector 117, unlike conventionalsystems which detect forward and side fluorescence and which use two ormore photodetectors. However, it will be understood that while anepi-illumination system is preferred, many of the aspects of thisinvention can be applied regardless of the type of optics system used.

In one embodiment, the epi-illumination instrument 417 comprises arectangular base 429 supporting a plurality of optical elements. Theseoptical elements are described below, with specific examples of relevantdimensions, focal lengths, and part numbers. As will be understood bythose skilled in the art, this information is exemplary only, andalternative optical elements can be used without departing from thescope of this invention.

Referring to FIGS. 28-34, the optical elements include a reflectingfilter 431 which reflects a collimated beam 25 of light from a laser orarc lamp 435, for example, through a conditioning lens assembly 437mounted in an opening 439 in a side wall 441 of a dichroic chamber 443extending up from the base 429. In this particular embodiment, theconditioning lens assembly 437 comprises a retaining ring 445, neutraldensity filter 447, cylindrical lens 449, lens holder 455 and jam nut457. The cylindrical lens 449 introduces a one-dimensional divergenceinto the beam 225 and directs it toward optical elements (describedbelow) which shape the beam to have a desired cross sectional shape 459,preferably generally elliptical. By way of example, the cylindrical lens449 may be a plano-convex lens having a focal length of 16 mm. A beamexpander (not shown) can optionally be installed in the instrument 417to allow adjustments to be made to the shape of the elliptical beam spot459.

The reflecting filter 431 is mounted by clips 461 on the angular face465 of a filter holder 463 which has openings 467 in it to permit thebeam 25 to reflect off the filter 431 toward the optics of theinstrument 417. The holder 463 is fastened to a linear stage 469 movablealong an X-axis 471 relative to an outrigger 473 secured to the base 429and dichroic chamber 443, the stage 469 being movable by suitable means475 (e.g., a micrometer) to precisely locate the holder 463 andreflecting filter 431 to reflect the beam 25 into the instrument 417 atthe proper location. A dichroic filter 477 is held by clips 479 on aframe 485 mounted in the dichroic chamber 443 and functions to reflectthe shaped beam 25 in a forward direction 487 along an axis 489 which,in this particular embodiment, corresponds to the longitudinal opticalaxis 419 of the instrument. The beam 25 passes through a focusing lensassembly 491 which focuses the beam 25 on the fluid stream 21 as a beamspot having the aforementioned generally elliptical shape 459 (FIG. 6)with the major axis of the ellipse extending generally perpendicular tothe direction of flow 227 of the stream 21. As each cell passes throughthe beam spot 459, the fluorescing dye (or other reporting agent) in thecell is activated to emit fluorescent light 31 (FIG. 23). In the case ofsperm cells stained with a DNA selective fluorescing dye, X cells havemore DNA than Y cells, include more fluorescing dye, and emit a strongersignal than Y cells (e.g., 3.8%), which provides a basis fordiscriminating and sorting cells, as will be described. The focusinglens assembly 491 includes, in one embodiment, a microscope adapter 501mounted in an opening 503 in a front wall 505 of the dichroic chamber443, a focusing barrel 507, a pair of lens mount barrels 509, and thelens 511 itself, which may be a 12.5 mm diameter, plano-convex lens witha focal length of 16 mm, available from Oriel Corporation as part number41209, and is anti-reflective coated for light having a wavelength inthe range of 340-550 nm. The lens 511 may be made of fused silica. Otherfocusing lenses may also be suitable, such as an infinity-correctedfluorescence microscope objective. The focusing lens assembly 491 has aconventional telescoping focus adjustment 515 to focus theelliptically-shaped beam spot 459 on the core 189 of the stream 21.

The outgoing fluorescent light 31 emitted by the cells as they passthrough the beam spot 459 is of a different (longer, due to the Stoke'sshift principle) wavelength than the incoming laser light 25. Some ofthe fluorescence emissions 31 are transmitted in a rearward direction513 along the incoming beam axis back through the focusing lens 511which collects and collimates the fluorescence emission 31. Thecollimated fluorescence emissions 517 pass in a rearward direction fromthe lens 511 to the dichroic filter 477, which transmits thefluorescence emission 517. By way of example, the dichroic filter 477may be a filter available from Omega Optical as part number XF2001,400DCLP.

The optics system 415 includes a filtering system 519 positionedrearward of the dichroic filter 477 along the optical axis 419 of theinstrument 417. In one embodiment, the filtering system 519 includes anemission filter 521 in a holder 523 mounted in an opening 525 in a backwall 527 of the dichroic chamber 443. The emission filter 521 attenuatesany laser light scatter or other undesired electromagnetic radiationthat is transmitted through the dichroic filter 477. By way of exampleand not limitation, the emission filter 521 can be a thin film,long-pass filter adapted to transmit more than 90% of light having awavelength greater than 408 nm, as is available from Omega Optical aspart number XF3097. An alignment pellicle assembly 529 is spacedrearwardly along the optical axis 419 from the emission filter. Thisassembly includes a slider 531 movable on a rail 533 extendinglongitudinally of the base 429 parallel to the longitudinal optical axis419 of the instrument 417, a filter holder 535 secured to the slider531, a pellicle filter element 539, and clips 541 for securing thepellicle filter element 539 to the filter holder 535 at an angle 543relative to the optical axis 419 of the instrument 417. The pelliclefilter element 539 has the same thickness as the dichroic filter 477 andfunctions to translate the collimated fluorescence emission 517 backonto the optical axis 419 of the instrument 417. Fasteners 545 extendingup through parallel slots 547 in the base 429 on opposite sides of therail 533 secure the slider 531 to the base 429 in the desired positionalong the optical axis 419. Spaced to the rear of the alignment pellicleassembly 529 is an aspheric lens 549 held by a holder 551 mounted in aframe 553 which is also slidable on the rail 533 and secured in selectedposition by suitable fasteners 557. The aspheric lens 549 focuses thecollimated fluorescence emission 517 onto a spatial filter, generallydesignated 559, which filters out reflection or emission from sourcesother than the cells to be analyzed. The aspheric lens 549 may be, forexample, a 12.5 mm diameter aspheric lens having a focal length of 15mm, as is available from Oriel Corporation. The lens 549 is preferablyanti-reflective coated for visible emission wavelengths but made of amaterial (e.g., flint glass) which further attenuates transmission oflaser light scatter.

As shown in FIG. 34, the spatial filter 559 comprises, in oneembodiment, a pair of aperture plates 561 releasably held by a frame 563mounted on the base 429 of the instrument 417. Each of the plates 561has a slit 567, 571 therein, one slit 567 preferably being generallyvertical and the other 571 preferably generally horizontal, thearrangement being such that the slits 567, 571 intersect to form anaperture 573. In one embodiment, the aperture 573 is generallyrectangular in shape and has a vertical dimension 575 of 100 microns anda horizontal dimension 577 of 500 microns. The size and shape of theaperture 573 may vary (or even be adjusted by changing aperture plates),so long as it functions to remove reflections and light from any sourceother than the collection volume 579. The frame 563 holding the apertureplates 561 preferably has two parts, namely, a plate holder 583 slidableon the rail 533 of the base 429 and secured in selected position byfasteners 587, and a backing member 589 for securing the aperture plates461 in position on the plate holder 583.

In one embodiment, the smaller (vertical) dimension 575 of the aperture573 in the spatial filter 559 is sized (or adjusted) to enable use of a“slit scanning” technique to evaluate the cell. This technique isdescribed in more detail in the “Focused Beam Spot” section of thisspecification.

Another embodiment of an epi-illumination optics system, generallydesignated 450, is shown in FIG. 35. This embodiment is substantiallythe same as the embodiment shown in FIGS. 28-34, except as noted. Onesignificant difference is that the dichroic filter 477 has been replacedwith a different dichroic filter 451 that transmits (rather thanreflects) the illumination beam 25 and reflects (rather than transmits)the fluorescent emissions 31. Also, because the fluorescence emissions31 are reflected by the dichroic filter 451 rather than transmitted,there is no need for an alignment pellicle 539 in this embodiment of anepi-illumination optics system 450. Thus, the epi-illumination system450 is just one example of how the optics system can be reconfigured ifdesired without departing from the scope of this invention.

Further, the cylindrical lens 449 is mounted on an adjustable mountingassembly 449A. The mounting assembly 449A allows two-axis translationalmovement of the cylindrical lens 449 in a plane perpendicular to theillumination beam 25. Releasable fasteners (e.g., screws (not shown))extend through slot-shaped holes 449B (only one of which is visible onFIG. 35). Release of the fasteners allows translational movement of thelens 449 in a first direction perpendicular to the beam 25. Similarfasteners (not shown) extend through slot-shaped holes 449C, allowingtranslational movement of the lens 449 in a second directionperpendicular to the first direction. This allows minor adjustment ofthe relative positions of the cylindrical lens 449 and beam 25 so thatthe intersection of the beam 25 and lens 449 can be moved across thesurface of the lens 449, thereby causing slight changes to the focusingprovided by the cylindrical lens 449. Once the lens 449 is in thedesired position, the fasteners can be tightened to hold it there.

Photodetector

The emitted fluorescence passing though the spatial filter 559 fallsupon a photodetector 117 fastened to a mounting plate 591 slidable onthe rail 533 of the base 429 at the rear of the epi-illuminationinstrument 417 and securable in fixed position by fasteners 595 (FIG.32). The photodetector 117 detects the fluorescent emissions 31 andconverts them into electrical signals which can be processed to analyzethe desired characteristics of the cells, as will be described in moredetail later. The photodetector 117 may be a conventional device, suchas a photodetector available from Hammamtsu. The photodetector 117preferably includes a preamplifier and PMT gain which is optimized foremission intensity produced by the epi-illumination system for theparticular stained cells being analyzed.

In general, the PMT gain is optimized when between about 200 and 2000volts are applied to the vacuum tube. In the case of detectingfluorescent emissions from Hoechst 33342, for instance, the PMT gain isoptimized when between about 400-800 volts are applied to the vacuumtube. One particularly desirable photodetector includes a PMT having aspectral range of 185-830 nm (530 nm peak), a 0.01 mA maximum averageanode current, a cathode radiant sensitivity of 70 mA/W typical, acathode luminous sensitivity of 140, μA/lm, anode luminous sensitivityof 300 A/lm, max anode dark current of 1 nA (0.1 nA typical), and a 1.4nanosecond risetime. The PMT is DC coupled amplifier demonstrating aflat gain to >37 MHz, having a 1 V peak output into a 50Ω load and arecovery time of less than 400 nanoseconds. It is also desirable for theamplifier to allow high voltage adjustment for compensation of PMTefficiency variations without decreasing the signal-to-noise ratio toless than 800 dB.

Angle of Beam Incidence

FIG. 36 schematically illustrates one desirable orientation of theintersection of the light beam and the fluid stream. Several points areof note. As shown, the light beam 25 is focused on the stream 21 at alocation 115 that is only a short distance 605 from the exit orifice 103of the nozzle 137, preferably less than 1.0 mm, or even inside thenozzle 137, so that the cells pass through the spot 459 while they arestill substantially in desired orientation, as previously described.This is particularly important for cells which are mobile in the fluidstream 21, including sperm cells.

Another point of note is that the beam 25 of this embodiment may bedirected toward the fluid stream 21 along a beam axis 609 whichintersects the fluid stream 21 at an angle of incidence A which isskewed (off 90 degrees) relative to a longitudinal axis of the fluidstream 21, as viewed from a side of the stream 21 (see FIG. 36). Whensorting certain particles, it has been found that better discriminationof the different types of particles may be obtained by illuminating thestream 21 at an angle of incidence other than 0°. Sperm nuclei, forinstance, are desirably illuminated at an angle of incidence A that isin the range of 5 to 45 degrees, more preferably in the range of 15 to30 degrees, and even more preferably in the range of 18 to 24 degrees.Other particles (e.g., live sperm cells) are easier to interrogate whenthe light beam 25 is generally perpendicular to the fluid stream 21(i.e., when angle A is about 0°). Thus, it is contemplated that angle Amay be any angle without departing from the scope of this invention.

The proper selection of angle A results in improved signal to noisediscrimination in certain particles and thus more accuratediscrimination based on different characteristics of those particles(e.g., sperm nuclei with X and Y chromosomes sperm cells). Thisimprovement may be due to a number of factors, including reduced laserlight scatter entering the focusing lens 511. Because the focused beamspot 459 is preferably wider than the stream 21, a diffraction patternis created at the intersection 115 of the beam 25 and the stream 21.When angle A is greater than about 12 degrees, the reflected diffractionpattern does not fall on the lens 511. Another factor may be that theskewed angle A allows the beam 25 to be focused very close to the nozzleorifice 103, so that the nozzle body 139 does not interfere with thelens 511. Relatedly, the cells are more uniformly aligned closer to thenozzle 137, so that focusing the beam spot 459 closer to the nozzle 137results in an improved signal. Further, the more “head on” profile ofthe cell presented to the lens 511 (beam 25) at the skewed angle Areduces the variation of total fluorescence intensity caused by anymisalignment of the cells. In this regard, in the case of sperm cells itis preferable that the beam 25 fall on one of the wide faces 207 of eachsperm cell 201, as discussed above, and that the nozzle 101 and opticssystem 109 be positioned to achieve this result.

While a skewed angle of incidence A is believed to be beneficial insorting some particles, it is contemplated that the angle ofintersection between the beam axis and the stream may be 90 degrees orany skewed angle without departing from the scope of this invention. Itis also expected that the optimal angle of incidence may vary widelydepending on the properties of the particular particles being analyzed.

Focused Beam Spot

Referring to FIG. 6, the focused beam spot of one embodiment is shown ashaving a generally elliptical (oval) shape 459 with a length L1 along amajor axis extending generally at right angles to the direction of fluidstream flow 227 and a width W1 along a minor axis extending generallyparallel to the direction of fluid stream flow 227. In one embodiment,the width W1 is less than the length of the head of the sperm cell 219,and even more preferably less than the length of the region 225containing the chromatic DNA mass of the cell, which in the case of abovine sperm cell 201 has a length of less than about 1μm. For a stream21 having sheath stream 191 that is about 60 μm in diameter and a corestream 189 containing bovine sperm cells 201, an exemplary length L1 isabout 80 μm and an exemplary width W1 is about 1.5 μm. By focusing thebeam spot 459 to a width W1 which is less than the length of the head205 of the sperm cell 201, or any other cell or particle being analyzed,and even more preferably less than the diameter of the DNA region 225 ofthe head 205 of the sperm cell 201, greater signal resolution isachieved, as will be understood by those familiar with “slit scanning”techniques. This is a technique by which a beam 25 is narrowed to have awidth less than the length of a cell (i.e., the dimension of the cell inthe direction of stream flow) so that as the cell moves through thenarrow beam, photon emissions 31 from the cell are measured over thelength of the cell, as will be discussed later. In this way, informationcan be obtained about variations in structure, including DNA material,along the length of the cell. The slit-scanning technique is alsohelpful in identifying “coincident” cells, that is, cells which areoverlapping or very close together.

As mentioned previously, slit scanning can also be carried out by sizingthe aperture 573 of the spatial filter 559 to have a vertical dimension575 such that only a portion of the light emitted from a cell,corresponding to a fraction of the cell length in the direction ofstream flow, passes through the aperture to the photodetector 117.Further, signal resolution can be optimized by adjusting the width ofthe beam and/or the size of the aperture of the spatial filter to worktogether to provide a beam spot that is suitably shaped for slitscanning.

One way to adjust the shape of the beam spot 459 is by changing to adifferent cylindrical lens and/or by making an adjustment to a beamexpander in the optics system 109. Further any method of shaping thebeam 25 to form an elliptically shaped beam spot 459 is contemplated asbeing within the scope of the present invention. Beam spots of othershapes and sizes may also be used and are contemplated as falling withinthe scope of this invention.

Sorting System

FIG. 2 illustrates an exemplary embodiment of the sorting system 119.The sorting system 119 comprises an electrostatic charging device 627for charging and/or not charging the droplets 33 depending on theclassification of the particles contained in the droplets 33 (e.g., theX/Y chromosome content of sperm cells), and a pair of electrostaticcharged deflector plates 629 for sorting the droplets 33 into differentgroups 123, 125, according to their charge. It is desirable to coat thedeflector plates 629 with a dull, low-emissive coating (e.g., epoxy orpaint) to limit light reflected or emitted by the deflector plates 629.The deflector plates 629 may be charged by any suitable power supply635. It is generally desirable for the electrical potential between thetwo fully charged deflector plates 629 to be in the range of 2000-4000volts. However, the electrical potential between the deflector plates629 may be anywhere between about 1000 and 6000 volts.

The charging device 627 comprises a charging element 631 having anopening 633 therein through which the stream 21 passes at a locationnear the droplet break-off location 107 (e.g., within five dropletlengths or closer). It is desirable to mount the charging element 631with a mechanism that facilitates adjustment of the position of thecharging element 631 with respect to the droplet break-off location 107.As shown in FIGS. 26 and 27, for example, the charging element 631 anddeflector plates 629 may be attached to an adjustable mounting assembly5001 that allows three-axis translation and tilt adjustment of thecharging element 631 and deflector plates 629 with respect to the nozzlesystem 101. For translation along an axis 5011 parallel to the stream21, the mounting assembly 5001 includes a board 5003 fastened to abacking 5005 by releasable fasteners 5007 passing through slots 5009 inthe board 5003, the slots 5009 being oriented generally parallel to axis5011. For translation in an axis 5013 perpendicular to the stream 21, asecond adjustment board 5015 is fastened to the first board 5003 byreleasable fasteners 5017 passing through slots 5019 in the secondadjustment board 5015, the slots 5019 being oriented generally parallelto axis 5013. The charging element 631 and deflector plates 629 aresecured to the second adjustment board 5015. Thus, by releasing thefasteners 5007 and/or 5017, one can adjust the position of the chargingelement 631 and deflector plates relative to the nozzle system 101 in aplane parallel to the fluid stream 21 and then tighten the fasteners5007 and/or 5017 to secure the mounting assembly 5001.

For translation along a third axis perpendicular to the first two axes5011, 5013, the backing 5005 is fastened to a fixed support 5021 byadjustable fasteners 5023 (e.g., threaded bolts screwed into tappedholes in the fixed support 5021). In one embodiment, each adjustablefastener 5023 passes through a spring 5025 positioned between thebacking 5005 and the fixed support 5021. The amount of compression ofany spring 5025 can be adjusted by tightening or loosening therespective fastener 5023. Adjusting the compression of all springs 5025in the same amount results in translation along the third axis. Themounting assembly 5001 can be tilted in virtually any direction bychanging the relative compression of one or more of the springs 5025with respect to one or more other springs 5025.

In this exemplary embodiment, the relative positions of the chargingelement 631 and deflector plates 629 remain fixed with respect to oneanother because they are all fastened to the same adjustment board 5015.This prevents adjustment of the mounting assembly 5001 from affectingalignment of the changing element 631 with respect to the deflectorplates 629.

The charging element 631 is connected to a suitable electrical circuit(e.g., a 90 volt selectively charging circuit) under the control of theprocessor 131 and coupled to a power supply for applying an electricalcharge to the charging element 631. The circuit is used to charge or notcharge the stream 21 immediately prior to the formation of a droplet 33at the break-off location 107 depending on whether the droplet 33contains a particle having the desired characteristics (e.g., at leastone live X-chromosome sperm cell). The charging element 631 ispositioned electrostatically near the stream 21 or near the droplets 33formed from the stream 21 for providing an electrical reference withrespect to the electrostatic polarity of the stream 21. The droplets 33carry the same charge as the stream 21 at the instant the droplet 33breaks from the stream 21. The charged or uncharged droplets 33 thenpass between the deflector plates 629 and are sorted by charge intocollection vessels 2207 of the collection system 2201. While sortingproduces two groups or populations of droplets 123, 125 in FIG. 2, theparticles may be separated into any number of populations from 1 to Nsorted by placing different charges on the droplets 33 in respectivegroups, any by supplying the appropriate number of collection vessels,each being positioned to collect a different population of droplets.

Automated Drop Delay Calibration

In the sorting system 119 described above, the processor 131 mustestimate the time it takes for a particle to move from the interrogationlocation 115 to the droplet break-off location 107 so that the charge(or lack of charge) to be applied to the droplet 33 containing thatparticle is applied when the particle is in the last attached droplet 33at the break-off location 107. If the delay setting used by theprocessor 131 is wrong, the droplets 33 will not be sorted according totheir contents. Similarly, if the application of electrical charges tothe droplets 33 is even slightly out of phase with droplet 33 formationthis can degrade sorting because none of the droplets 33 will be fullycharged and droplets 33 that are supposed to have neutral charge willcarry a small positive or negative electrical charge. This will alterthe paths of the droplets 33 through the electric field between thedeflection plates 629.

The best way to verify that the processor 131 is using the appropriatedelay setting or to adjust the drop delay setting (i.e., calibrate thesystem's 9 drop delay setting), is to sort a number of droplets 33 andexamine the results. By incrementally varying the delay setting andmonitoring the results, one can select the optimal delay setting.Traditionally, this sort calibration is performed manually. Recently,automated calibration systems have been designed to sample or examinethe contents of the droplets in the sorted droplet streams andautomatically adjust the delay setting without human intervention. Forexample U.S. Pat. No. 6,372,506 (Norton) and U.S. Pat. No. 5,643,796(van den Engh), which are hereby incorporated by reference, bothdisclose automated sort calibration systems. The purported advantages ofthese systems are that they are less labor intensive and are capable ofverifying the delay setting throughout the sorting process rather thanjust during initial set up. The drawbacks are that they are cumbersomeand take up valuable space unnecessarily.

(i) Epi-Illumination Sensors

Referring to FIG. 37, an automated continuous calibration system 4201 ofthe present invention for a fluorescence activated droplet sortingcytometry system comprises one or more epi-illumination sensors 4203positioned to sense the contents of droplets 33 to verify the delaysetting for droplet charging. Referring to FIG. 38, eachepi-illumination sensor includes a light source (not shown), a fiberoptic cable 4205, a dichroic filter 4207, a lens system 4209, aphotodetector 4213, and a control system. In one exemplary embodiment,the processor 131 serves as the control system, but other processors orcontrols could be used instead.

The light source may be a low-power solid state laser dedicated solelyto the automated calibration system 4201. Alternatively, a beam splitter(not shown) may be used to divert a portion (e.g., about 5%) of theenergy in the beam 25 used for interrogation of particles in the fluidstream 21 to one or more epi-illumination sensors 4203. Similarly, thefiber optic cable 4209 can be positioned in a beam stop 4215 (FIG. 26)to gather light from beam 25 after it passes through the interrogationlocation 115. The light from the light source must include light havinga wavelength capable of exciting fluorescent molecules in the particlesbeing sorted, thereby causing fluorescence emissions 4211 from theparticles. If the particles are stained with Hoechst 33342, forinstance, the light source can provide light having a wavelength ofabout 350 nm, about 407 nm or any other wavelength capable of excitingthe Hoechst 33342 molecules.

The fiber optic cable 4205 extends from the light source to a locationdownstream of the interrogation location 115. For example, in theexemplary embodiment the fiber optic cable 4205 leads to a locationadjacent the trajectory of one of the droplet streams as it movesthrough the electric field between the deflector plates 629. Thedichroic filter 4207 is positioned in front of the end of the fiberoptic cable 4205. The dichroic filter 4207 transmits light having thespectral characteristics of the light conducted by fiber optic cable4205, but reflects light having the spectral characteristics of thefluorescence emissions 4211. Thus, the dichroic filter 4207 may have thesame specifications as the dichroic filter 477 described above inconnection with the epi-illumination optics instrument 417. The focallength of the lens system 4209 is selected based on the expecteddistance of the sensor 4203 from the droplets 33 so that theillumination/detection volume of each sensor 4203 is about equal to thevolume of the droplets 33.

Referring to the exemplary embodiment shown in FIG. 37, anepi-illumination sensor 4203 is positioned adjacent the trajectory ofeach of the three sorted droplet streams 4225, 4227, 4229 to sense thecontents of droplets 33 in a respective stream. The cytometer system 9includes an electrically insulated support 4221 for mounting the twodeflection plates 629. The support has three holes 4223, one adjacentthe trajectory of each sorted droplet stream 4225, 4227, 4229. Anepi-illumination sensor 4203 is positioned at each hole 4223 to observedroplets 33 in one of the droplet streams 4225, 4227, 4229 through therespective hole 4223. This compact configuration takes up relativelylittle space and keeps components of the calibration system 4201 out ofthe way, providing better access to other parts of the cytometer 9.

If a droplet containing a fluorescent particle passes through theillumination/detection volume of the sensor 4203, this will result in aflash of fluorescence emissions 4211, some of which will be collected bythe lens system 4209 and reflected off from the dichroic filter 4207 tothe photodetector 4213. Signals from the photodetector 4213 are providedto the processor 131. Based on the signals received from thephotodetectors 4213, the processor 131 can determine the contents of thedroplets 33 in each of the sorted droplet streams 4225, 4227, 4229.

If a sensor 4203 fails to detect a flash of fluorescence emission 4211when the processor 131 expects a droplet 33 containing a fluorescentparticle to pass by that sensor 4203, the processor 131 can use thatinformation to adjust the delay setting or adjust the location of thedroplet break-off location 107. Likewise, the processor 131 can make anadjustment if a sensor 4203 detects a fluorescent emission 4211 when theprocessor 131 does not expect a droplet 33 containing a particle to bepassing by the sensor 4203. Furthermore, the processor can compare therelative frequency of fluorescent emissions 4211 from the sorted streams4225, 4227, 4229 to see if the frequency of detected fluorescentemissions 4211 matches the expected frequency. The processor 131 canalso adjust the amplitude of the charge applied to the charging element631 to increase or decrease the amount by which a sorted stream 4225,4229 is deflected to maximize the intensity of the detected fluorescenceemissions 4211. This will maintain the alignment of the trajectory ofthe deflected droplet streams 4225, 4229 so the droplets pass directlythrough the collection volume of the epi-illumination sensor. Becausethe sensors 4203 are positioned to observe the streams 4225, 4227, 4229as they move through the electrical field between the deflector plates629, the calibration system has a shorter response time than it would ifit observed the streams 4225, 4227, 4229 in the freefall area downstreamof the deflection plates.

(ii) Empty Droplet Test Stream

One sensitive indication of the quality of the calibration can bearranged by creating and monitoring a calibration test stream thatcontains substantially only empty droplets 33. Referring to the sortcalibration system 4201 shown FIG. 37, droplets 33 containing desiredparticles are sorted into stream 4225 and droplets 33 containing anyother particles and most of the empty droplets 33 are sorted into stream4229 (i.e., the waste stream). The test stream 4227 is created byapplying a neutral charge to at least a fraction (e.g., 1 out of every10) of the empty droplets 33. Many droplets 33 that are considered“empty” for traditional sorting purposes are actually droplets 33 forwhich there is a low probability that the droplet 33 contains aparticle, based on the arrival time of particles at the interrogationlocation 115 and estimated droplet formation boundaries in the fluidstream 21. These “empty” droplets should not be sorted into the teststream 4227 because this would inevitably result in detection of someparticles in the test stream 4227.

Instead, for the test stream 4227 the processor 131 should select onlydroplets 33 that the processor 131 believes have substantially zeroprobability of containing a particle in order to create a substantiallyparticle-free test stream 4227. The probability that any randomlyselected droplet 33 contains a cell is known and is approximately theaverage cell analysis rate divided by the droplet generation rate. Thismeans that by monitoring the rate of mis-sorts in the test stream 4227it is possible to estimate fractional adjustment of the phaserelationship of droplet charging needed to match the phase of droplet 33formation. For example the processor 131 may select droplets that itestimates have about 15% or lower probability of containing a particle,about 10% or lower probability of containing a particle, about 5% orlower probability of containing a particle, about 1% or lowerprobability of containing a particle, about 0.1% or lower probability ofcontaining a particle, about 0.01% or lower probability of containing aparticle, about 0.001% or lower probability of containing a particle, orabout 0.0001% or lower probability of containing a particle. Theprobabilistic cutoff for substantially zero probability may be selectedbased on sort-speed, tolerance for impurity, or other sort parameters,with the cutoff including higher probabilities that a droplet willinclude a particle for high-speed sorting or when there is moretolerance for impurity.

Failure of the processor 131 to create a substantially particle-freetest stream 4227 (i.e., a test stream 4227 in which the ratio ofdroplets 33 containing particles to the total number of droplets 33agrees with the probabilistic cutoff used to select droplets 33 for thetest stream 4227), as indicated by detection of more than a thresholdnumber of droplets 33 containing particles in the test stream 4227, is adefinitive indication of sub-optimal sorting and prompts the processor131 to adjust the drop delay setting. The threshold level is determinedin relation to the probabilistic cutoff used to select droplets 33 forthe test stream 4227 and the total number of droplets 33 selected forthe test stream 4227. Ideally, some droplets 33 can be selected for thetest stream 4227 even though one or more particles in the fluid stream21 are relatively close to an estimated drop formation boundary for therespective droplet 33 to make the system 4201 more sensitive to slightlysub-optimal drop delay settings.

Of course, the sort calibration system could apply a non-neutral chargeto and deflect droplets selected for the test stream, without departingfrom the scope of this invention. The relative order of the streams4225, 4227, 4229 could also be rearranged without departing from thescope of this invention, although interposing the test stream 4227between the waste stream 4225 and the stream of desired particles 4229(as shown in the exemplary embodiment) reduces the risk of crossovercontamination of the sorted sample by the waste stream. Further, if theparticles do not emit fluorescent light, different sensors can be usedto detect any scattered light caused by particles in the test streamwithout departing from the scope of this invention.

(iii) Impact of Sort Calibration System

In one embodiment of the invention, the automated calibration system4201 is operable to automatically determine and set the phaserelationship between droplet formation and droplet charging to withinabout 5% of the optimal phase (i.e., within +/−about 18 degrees. Inanother embodiment the system 4201 is operable to automaticallydetermine and set the phase relationship to within about 1% of theoptimal phase (i.e., within +/−about 3.6 degrees)). In anotherembodiment, the calibration system 4201 is operable to continuouslymonitor a high-speed droplet sorting system and automatically maintainthe phase relationship within about 10% of the optimal phase (i.e.,within +/−about 36 degrees). In still another embodiment, the system4201 is operable to continuously monitor a high-speed droplet sortingsystem and automatically maintain the phase relationship without about3% of the optimal phase (i.e., within +/−10.8 degrees).

Sort System Fault Correction

From time to time, a droplet 33 will stray from its normal trajectoryand hit the charging element 631 or the deflector plates 629. If one ormore droplets 33 hit the charging element 631, the charging element 631may not be able to charge droplets 33 properly. Further, the normaldroplet 33 trajectory through the charging element 631 can becomeobstructed causing even more droplets 33 to accumulate on the chargingelement 631. Also, if stray droplets 33 strike a deflector plate 629,they can distort or otherwise disrupt electrical field lines between thedeflector plates 629, thereby changing the trajectory of the sorteddroplet steams 123, 125.

Thus, it is desirable to have a debris removal system to remove debrisfrom the charging element 631 and/or the deflector plates 629. In oneexemplary embodiment, shown FIGS. 26 and 27, the system 9 includes adebris removal system 5047 for the charging element 631 and a debrisremoval system 5049 for the deflector plates 629.

Referring to FIG. 27, the charging element 631 is held in position by asupport 5051 secured to board 5015 of the adjustable mounting assembly5001. A vacuum passage 5053 (shown in phantom) extends through thesupport 5051 to an opening 5057 adjacent the charging element 631. Thevacuum passage 5053 is connected to a suitable vacuum source (not shown)by a vacuum line 5055 attached to a fitting 5058 on the support 5051.Suitable controls are provided for selectively applying a vacuum in thepassage 5053 to vacuum any undesired material (e.g., stray droplets 33)off the charging element 631 and restore proper function of the chargingelement 631.

Relatedly, as shown in FIG. 27, a manifold 5061 fastened to the mountingassembly 5001 has a network of air passages 5063 therein (shown inphantom) connected via an air line 5059 and fitting 5065 to a source ofcompressed air or other gas (not shown). The passages 5063 have openings5064 positioned along a side 5066 of each deflector plate 629 and theportions 5067 of the passages 5063 leading to the openings 5065 areoriented so compressed air blown through the manifold 5061 will clearany stray droplets 33 or other debris off the deflector plates 629. Anymaterial blown off the deflector plates 629 will hit a cover panel (notshown) and drain into a suitable waste collection device (not shown).

In one embodiment, if the processor or other sensor determines thatstray droplets 33 have hit the charging element 631 or deflector plates629, as indicated by the sort calibration system described above forexample, the processor can automatically initiate a fault correctionprocedure or mode, which can include applying a vacuum to passage 5053to vacuum material from the charging element 631 and/or sendingcompressed gas through passages 5067 to blow material off the deflectorplates 629.

Protection of Sorted Sample During Fault Mode

One embodiment of the system 9 also includes a contamination preventionmechanism 4041 (FIG. 26), which can be activated by the processor 131 tolimit or prevent contamination of the sorted sample any time the sortingsystem is in the fault correction mode. The contamination preventionmechanism includes a pneumatic actuator 4043 operable to selectivelymove a swing arm 4045 between a shielding position (shown FIG. 26) and anon-shielding position (not shown). In the shielding position, the end4047 of the swing arm 4045 covers the opening of the collection vessel4033, thereby preventing collection of droplets 33 by the collectionvessel 4033. In the non-shielding position, the collection vessel 4033is uncovered. Normally, the swing arm 4045 is in the non-shieldingposition, but the processor 131 causes the actuator 4043 to move theswing arm 4045 into the shielding position any time the processor 131determines that there is a risk of contamination (e.g., the nozzlesystem 101 becomes clogged, the droplet break-off location 107 becomesunstable, or stray droplets 33 have hit the charging element 631 ordeflector plates 629). The end 4047 of the swing arm 4045 istrough-shaped to drain any fluid collected by the swing arm 4045 intothe waste container 4035.

Fluid Delivery System

The system 1 described above is capable of effectively producingquantities of particles (e.g., X-sperm cells) sorted by selectedcharacteristics. The rate of production can be increased or decreased byvarying the rates at which the fluid delivery system 15 (FIG. 2)delivers carrier fluid 17 and sheath fluid 19 to the nozzle 137. In oneembodiment, the fluid delivery system includes a syringe pump 645, oneexample of such a pump being MICROLAB® Model PSD/3 available fromHamilton Company. The pump 645 is operable to deliver carrier fluid 17to the nozzle 137 at a rate of about 20 μl/min. In general, the pump 645should be operable to deliver sample fluid 17 to the nozzle 137 at arate in the range of 10-50 μl/min. The pump 645 is connected by a flowline 647 to the supply 3 of carrier fluid 17, which may be a suitablevessel 649 containing a volume of material to be analyzed and sorted.Where the temperature of the particles being analyzed is a factor, as inthe case of sperm cells, for example, the temperature of the vessel 649may be controlled by a suitable temperature control system, such asheating/cooling bath (not shown). The syringe pump 645 is movablethrough an intake stroke to aspirate carrier fluid from the supplyvessel and through a discharge stroke to dispense carrier fluid 17through a supply line 651 to the injection needle 157 of the nozzlesystem 101. The pump 645 is preferably driven by a variable speed motor(not shown) under the control of the processor 131. By way of example,the pump 645 may be driven by a stepper motor which operates atselectively variable rates to pump carrier fluid 17 to the needle 159 atrates necessary to obtain the desired throughput. Other types of fluiddelivery devices can be used instead of a syringe pump. To provide justone example, the vessel 649 can be pressurized by a pressurized gassource without departing from the scope of the invention. Furthermore,it is desirable to keep the lines 647, 651 as short as is practicallypossible because the line environment is not conducive to the health ofsensitive cells (e.g., sperm cells) that may be in the carrier fluid 17.

The supply 7 of sheath fluid 19 comprises a second vessel 661, e.g., atank in FIG. 2, holding an appropriate volume of sheath fluid 19connected to the radial bore 173 in the flow body 133 of the nozzlesystem 101 by a supply line 667 having a control valve 669 therein. Inthe embodiment of FIG. 1, the sheath fluid vessel 661 is pressurized bya gas pressure system 671 comprising a source 675 of pressurized gas(e.g., air or other gas, such as nitrogen) communicating with the tank661 via an air line 679 having a regulator 681 in it for controlling thepressure supplied to the tank 661. A two-way valve 683 in the air line679 is movable between a first position establishing communicationbetween the tank 661 and the gas source 675 and a second positionventing the tank 661. The gas pressure regulator 681 is a conventionalregulator preferably under the control of the processor 131. Bycontrolling the tank 661 pressure, the pressure at which sheath fluid 19is delivered to the flow body 133 may also be controlled. This pressuremay range from 16 to 100 psi, more preferably from 10 to 50 psi, evenmore preferably 15 to 40 psi, and even more preferably from about 20 to30 psi. The pressure at which the sheath fluid 19 is supplied to theflow body 133 can be controlled in other ways without departing from thescope of the invention.

In one embodiment, shown FIG. 26 the fluid delivery system 15, includesa sheath fluid tank (not shown) and a sample station 4051. The samplestation includes a two-part pressure container 4053 adapted to hold asample tube 4055. The bottom section 4057 of the pressure container ismoveable up and down relative to the upper section 4059 of the pressurecontainer 4053 between an open position (shown FIG. 26), in which thesample tube 4055 may be loaded or unloaded, and a closed position (notshown) in which the two parts 4057, 4059 of the pressure container 4053come together to form a seal to contain pressurized gas used to pumpcarrier fluid 17 from the sample tube 4055 to the nozzle system 101.

When the pressure container is open a spring-biased swing arm 4071 movesto a position beneath the line 651 that delivers carrier fluid 17 to thenozzle system 101 (See also FIG. 119 #4071′). The swing arm 4071 istrough-shaped and adapted to collect fluid backflushed through the line651 and to drain the backflushed fluid to the waste container throughport 4073. As the pressure container 4053 moves from its open positionto its closed position, a cam plate 4075 attached to the bottom section4057 of the pressure container 4053 moves the swing arm 4071 against itsspring bias to clear the area between the two sections 4057, 4059 andallow the pressure container 4053 to close.

Control

Referring again to FIG. 2, the microprocessor 131 (or other digital oranalog control and/or processor, or combinations thereof) controls theoperation of the system 1. As noted below with regard to FIG. 39, themicroprocessor may be implemented as a system control processor and fourprocessors for handling signal processing. Alternatively, some or allfunctions may be integrated into one or more processors. For example,the system control microprocessor (see FIG. 36) may be implemented byusing one of the four signal processing processors. In addition, asnoted below, the signal processing may be implemented by an analogcircuit (e.g., an analog cell analyzer as shown in FIG. 39) or acombination of analog and digital circuitry.

The microprocessor 131 provides output signals to control the fluiddelivery system 15 (noted below) in response to input signals receivedfrom the epi-illumination system 415, provides output signals to controlthe transducers 105 in response to input signals received from thebreak-off sensors 389, and provides output signals to control thesorting system 119 (noted below) in response to input signals receivedfrom the epi-illumination system 415. The microprocessor 131 may provideoutput signals to other parts of the cytometry system 9 as notedelsewhere herein. Further, the microprocessor 131 may be adapted toprocess information and provide output signals in real time. Broadlyspeaking, the term “real time” refers to operations in which theoperation of the processor 131 matches the human perception of time orthose in which the rate of the operation of the processor 131 matchesthe rate of relevant physical or external processes. In one context, theterm “real time” can indicate that the system reacts to events beforethe events become obsolete.

In general, electrical signals from the epi-illumination system 415 areconverted to digital information by an A/D converter 689 which suppliesthe corresponding digital information to the microprocessor 131. Inresponse to the information, the microprocessor 131 controls a sortingsystem 119 and a fluid delivery system 15, both described above.

The electrical signals output from the photodetector 117 of theepi-illumination system 415 are time-varying analog voltage signalsindicative of the amplitude of the emitted fluorescence 31 at anyinstant in time generated by each cell as it is illuminated by the laserbeam 25. Thus, the analog signals (also referred to as analog output)are in the shape of time-varying waveform pulses 497 as illustratedschematically in FIGS. 52 and 53. In general a waveform pulse 497 isdefined as a waveform or a portion of a waveform containing one or morepulses or some portion of a pulse. Thus, the amplitude of each waveformpulse 497 at any instant in time represents the relative rate of photonemission 31 of each cell at that instant in time as the cell passesthrough the laser beam 25. X chromosome bovine sperm cells have a higherDNA content than Y chromosome bovine sperm cells (e.g., about 3.8%). Asa result, live X cells labeled with a fluorescent stain as noted abovewill produce a different waveform pulse 497 than pulses from any otherlabeled cells. By analyzing the pulses 497 as noted below (see SignalProcessing, Slit Scanning, and Critical Slope Difference), each cell canbe identified as an X cell or not identified as an X cell (˜X). Ingeneral, as used herein, X cells refers to live X cells, Y cells refersto live Y cells and ˜X cells refers to the combination of live Y cellsand cells which otherwise produce a detectable fluorescence emission 31but which cannot be identified with a reasonable probability as beinglive X cells.

The timing of each waveform pulse 497 indicates the position of eachcell in the stream 21. Since the rate at which the sheath fluid 19 isbeing delivered through the nozzle 137 remains constant, and since thedistance d (in FIG. 25) between the nozzle 137 and the droplet break-offlocation 107 is known, the position of each droplet 33 is known and thecells, if any, within each droplet 33 are known. Thus, themicroprocessor 131 can calculate the instant at which each formingdroplet passes through the charging collar 631 and can control thepolarity of the collar 631 and thus control whether a droplet 33 ischarged for deflection by the charging elements 631 of the sortingsystem 119. Since the microprocessor 131 knows the droplet formationrate and identifies the cells within a droplet as X or ˜X, themicroprocessor 131 knows the cell content of each droplet 33 and keepstrack of (or enumerates) the number of cells in each population 123,125. Depending on the sort strategy, see below, the microprocessor 131determines which droplets 33 are charged for deflection and whichdroplets 33 are not charged so that they are not deflected.

Signal Processing

A. Digital Sampling Introduction

As previously described, the interaction between the laser beam 25 andthe particle produce a “pulsed” photon emission 31 (e.g., a fluorescenceemission) that is captured by the collection lens 511 of the opticssystem 109 and delivered to a photodetector 117. The photodetector 117converts the photon energy at any instant in time to an analog voltageoutput of time-varying amplitude. This output is a series of waveformpulses 497 (FIGS. 43 and 44) which contain many features that can beused to discriminate among populations of particles.

Among these features are the total photon emission, the rate of photonemission as a function of the particle's spatial transit through thelaser beam, the maximum rate of photon emission during the transit, theaverage rate of photon emission during the transit, and the timerequired for transit. The combination of laser beam geometry 459,particle size, distribution of the emission source through the particlevolume and particle velocity determine the frequency spectrum ofwaveform pulse 497. For the system 1 used with bovine semen describedpreviously it has been determined that each cell 201 produces a waveformpulse 497 of between 800 ns and 1200 ns in duration. It has also beendetermined that as a function of frequency, more than 97% of the powerin the waveform pulse 497 is delivered at frequencies below 30 MHz. Thisfrequency spectrum will be discussed later as it related to the Nyquistsampling theorem. Taken together these waveform pulses 497 form anoutput signal 701 from the photodetector 117 that is a continuous, timevarying, signal that represents the transit of the particle streamthrough the apparatus. In addition to features of individual pulses thatare used to discriminate among populations, the time varying signalprovides a precise record as to the relative spacing (time and position)among the individual particles that pass through the apparatus andrelative velocity of the particles moving through the apparatus. Thisprecise time, position and velocity record can be synchronized with thedroplet generation clock signals 703 as shown in FIG. 44 to determinewhich particles are members of a particular droplet 33 formed by thedroplet generation apparatus 105. This information can be used as thebasis for determining “coincidence” or the occurrence of a desired andundesired particle in a single droplet 33. The ability to accuratelydetermine the number and classification of each particle in a droplet 33allows for accurate, efficient sorting.

Digital signal processing 705 as illustrated in FIG. 72 may be employedto analyze detection of fluorescence pulses 31 as indicated bysynchronously sampled output signals 701 from the photodetector 117.This processing would be implemented in pulse analysis softwareemploying instructions and/or algorithms, as noted herein. Thetime-varying analog output signal 701 of the photodetector 117 isprovided to an A/D (analog/digital) converter 689 which synchronouslysamples it. Synchronously sampling means sampling to produce digitalinformation corresponding to the analog output. Synchronously samplingis also referred to as continuously sampling or streaming acquisition.As noted below, the sampling rate depends on the frequency spectrum ofthe analog output.

Converter 689 provides an output including digital information 707 whichis provided to the microprocessor 131 or other digital analysis devicewhich executes the pulse analysis software to analyze the digitalinformation 707. In general, the pulse analysis software would includedigital pulse detection HH3, pulse feature extraction HH4 and pulsediscrimination HH7.

B. Sampling Frequency & Signal Frequency Spectrum

The signal output 701 from the PMT 117 is captured by a high speedanalog to digital converter 689 (ADC) that samples the output 701continuously at a frequency of 105 MHz. It is well understood that whensampling a time varying signal it is necessary for the samplingfrequency to be at least twice the maximum frequency contained in thesignal being sampled. This is known as the Nyquist sampling theorem. Forthis reason the output signal 701 from the PMT 117 is first sent througha 40 MHz low-pass filter 854 (see FIG. 39) to ensure that the maximumfrequency contained in the signal 701 is under the 52.5 MHz limitimposed by the sampling rate. It is important to note that the optical109, fluidic 15 and detection systems of the apparatus 1 have been tunedto produce a pulse waveform 497 having optimum frequency characteristicsfor sampling at the 105 MHz rate. The sampling rate may be variedbetween about 25 and 200 MHz without departing from the scope of thepresent invention.

C. Pulse Processing

Pulse processing takes place in four (4) TigerSharc DSP processors thatshare memory and are connected to one another by high-speed parallelports. As illustrated in FIG. 39, the four processors are: 1) a datamanagement processor 863 which receives data from a high-speed ADC 689which digitizes the output signals 701 from the photodetector 117; 2) apulse detection processor 865 which detects the waveform pulses 497represented by the digital information; 3) a feature extraction anddiscrimination processor 867 which extracts features from the detectedpulses 497 and discriminates the pulses 497 based on the extractedfeatures; and 4) a sort processor 873 which determines a sortclassification for each pulse 497 based on the extracted features andthe discrimination, which determines sort decisions for thecorresponding cells and droplets 33 and which is synchronized withdroplet formation 105. In general a processor 863, 865, 867, 873completes a task and sets a “flag” so that companion processors knowthere is data available to process.

Each processor 863, 865, 867, 873 runs independently of the others,maximizing the overall throughput because they do not interrupt eachother. Thus, any processor 863, 865, 867, 873 may be capable ofperforming any function and one or more processors or functions may becombined into a single processor or spread out over a plurality ofprocessors. The processor 863, 865, 867, 873 labels as used above andthis application are used for convenience only and are not intended tobe limiting in any way.

All four processors 863, 865, 867, 873 are linked to a DSP board SDRAM851 for exchanging information and are linked to a processorinput/output (I/O) 857 for synchronization and communication with aperipheral I/O bus 859 connected to the PC 735 and the sort pulsegenerator 861. The processor I/O 857 may be implemented by two or moreSharcFIN I/O processors connected by a communication link. Sort signals853 are provided to the PC 735 via the peripheral I/O bus 857 and areused to control the sort pulse generator 861 controlling the charging ofdroplets 33.

The processor I/O 857 receives the output 707 from the analog/digitalconverter (ADC) 689, e.g., Bitware Corp. 105 MHz/2-channel, 14 bitcapable of 105 MHz/1-channel sustained. The ADC 689 is connected to thephotodetector 117 output for converting its time varying analog outputsignals 701 into digital information 707 and is also connected to an I/Oboard SDRAM 855 for storing the blocks of digital information from theADC 689.

In general, the analog output signals 701 from the photodetector 117 areindicative of characteristic A or characteristic B (e.g., X or ˜X). TheA/D converter 689 converts the analog output signals 701 from thephotodetector 117 of the flow cytometry system 1 into correspondingdigital information 707. The processors 863,865, 867,873 analyze andclassify the digital information 707 and provide a sorting signal to thesorting system 119 as a function of the detected and classified digitalinformation.

D. Data Acquisition

As previously stated, the signal output 701 from the photodetector 117is captured by a high speed analog to digital converter (ADC) 689 thatsamples the output continuously at a frequency of 105 MHz. Data (digitalinformation 707) are transferred immediately into high-speed memoryblocks (I/O Board SDRAM) 855 which serve to buffer the incoming data.These memory blocks 855 are organized in a manner to maintain theintegrity and sequence of the data stream 707. These memory blocks 855are also accessible by the digital signal processing (DSP) processors863, 865, 867, 873 by direct memory access (DMA). In this manner theprocessors 863, 865, 867, 873 can access the incoming data 707 withoutinterrupting the ADC 689. This facilitates efficient transfer of data707 to these processors 863, 865, 867, 873 for feature extraction,analysis and sort classification. Throughout this process, the datamanagement processor 863 keeps the pulse samples 707 in order and timeindexed (relative to the master clock 737, which is 128 times thedroplet 33 frequency) to preserve their reference to “real time” or theactual time that the cell passed through the laser beam 25. The ADC 689ping-pongs back and forth between two inputs, continuously sampling thetime varying analog output signals 701 including the waveform pulses 497and converting them into digital information 707 which is provided inblocks 855 to the I/O Board SDRAM under the control of the datamanagement processor 863. Processor 863 assembles the information 707into a continuous stream.

E. Initializing Detection Parameters

In order to effectively distinguish over background noise, the digitalpulse detection software 747 should be provided with informationindicating signal background second order statistics, i.e. knowledge ofthe behavior of the output voltage signal 701 from the photodetector 117when there is no fluorescence pulse 497. These statistics can be learnedby software for initializing detection parameters 741 in an unsupervisedmanner during the initialization period immediately following startup ofthe system 1. In general, a pulse may be defined as 2 or 3 standarddeviations from the background level.

Due to the possibility that introduction of the carrier fluid 17 intothe sheath fluid stream 191 may cause a change in backgroundfluorescence emission, the carrier fluid 17 should be present for theinitialization of the detection parameters. Simple computation of thesecond order statistics of a time sequence of output voltage signalvalues may overestimate the standard deviation of the background (due tothe possible presence of fluorescence pulses 497 in the sequence). Aniterative procedure is therefore preferred to gradually eliminate thiseffect. The pulse detection software 747 accomplishes this by computingthe statistics of the total signal 701 (background+pulses), using thesevalues to apply pulse detection logic, re-computing the signalstatistics without samples detected to be within pulses, and repeatingthis procedure until the background statistic estimates converge (or afixed maximum number of iterations occurs). By evaluating the backgroundwith cells present, a more accurate indication of the expected correctpulse 497 amplitude can be determined. Table III summarizes thedetection initialization procedure for determining detection parametersfor use by the pulse detection software.

TABLE III Initialization, of pulse detection algorithm parameters.Algorithm:  Initializing detection narameters Input:  vector of floatsPMTvolts; float statWindow Size, integer  maxlterations Output:  floatbckgrndMean; float bckgrndSTD Procedure: 1. Initialize background vectorbckgrnd to last statWindowSize samples of PMTvolts vector andnumlterations, lastSample- Mean, and lastSampleSTD to zero:   bckgrnd =PMTvolts[1 to statWindowSize]   lastSampleMean = 0   lastSampleSTD = 0  numlterations = 0 2. Compute sample mean and sample standard deviationof bckgrnd and increment iteration counter:   ${sampleMean} = \frac{{sum}({bckgrnd})}{statWinowSize}$   $\begin{matrix}{{sampleSTD} = \frac{\left. {{sum}\left( {{bckgrnd} - {sampleMean}} \right)}^{2} \right)}{statWindowSize}} \\{{numlterations} = {{numlterations} + 1}}\end{matrix}$ 3. Check for convergence or exceeding maximum number ofiterations:   exitFlag = ((sample  Mean − last  sampleMean < eps⋀(sampleStd − lastsampleStd < eps⋁(numlteration > maxIterations)If exitFlag is true, go to step 6 (else continue with step 4). 4. Applypulse detection algorithm, obtaining vectors of pulse samples and newestimate of background samples:   [pulse,bckgrnd] =pulse_detect(bckgrnd,sampleMean,   sample-STD) 5. Record statisticsestimates from this iteration and repeat          lastSampleMean =sampleMean   lastSampleSTD = sampleSTD Go to step 2. 6. Set backgroundstatistics estimates to sample statistics and exit:   bckgrndMean =sampleMean   bckgrndSTD = sampleSTD

In general, the A/D converter 689 converts the analog output signals 701from the photodetector 117 into corresponding digital information 707indicative of characteristic A or characteristic B (e.g., X or ˜X). Thedigital signal processor 865 determines background characteristics ofthe time-varying output signals 701 from the digital information 707corresponding thereto, detects waveform pulses 497 from the digitalinformation 707 as a function of the determined backgroundcharacteristics, and provides a sorting signal 853 to the sorting system119 as a function of the detected pulses 497.

F. Initial Discrimination Parameters

Similar to the detection parameters (and subsequent to theirinitialization as shown in Table III), parameters for use in adiscrimination algorithm may be initialized in an unsupervised fashion.Unlike the detection algorithm parameters, however, an iterativeprocedure is not necessary. In this case, software for initializing thediscrimination parameters 745 detects a preset number (e.g., 100,000) offluorescence pulses 497, computes the features to be used fordiscrimination for each detected pulse 497, and uses a clusteringprocedure (see Table IV for a summary of candidate clusteringprocedures) to assign these pulses 497 to populations of interest (e.g.X, ˜X).

TABLE IV Summary of clustering approaches being considered for use indiscrimination algorithm parameter initialization. Algorithm NameAlgorithm Approach k-Means Iterative (local) minimization of sum ofsquared distance (Euclidean or Mahalanobis) between points within eachpopulation [1] Fuzzy k-Means Expectation-Maximization of (Gaussian)mixture model [2] Agglomerative Merging of “nearest” clusters (startingwith each Hierarchical data point as its own cluster) until desirednumber of clusters is reached. Various measures for determination of“nearest” clusters include distance between closest points, distancebetween furthest points, distance between cluster means, and averagedistance between points. [1]

FIG. 73 contains an example of the results of application of a k-meansclustering procedure to define population 1 and population 2 based onstatistics of distribution. The second order statistics of thesepopulations are then used to set the parameters necessary fordiscrimination (the coefficients of a 1^(st) or 2^(nd) order polynomialdecision function). Table V summarizes the discrimination initializationprocedure.

TABLE V Initialization of discrimination algorithm parameters.Algorithm: Initializing discrimination parameters Input: Matrix offloats detectedPulseData, vector of floats popPriorProbabilities Output:For each class population i: matrix of floats W_(i), vector of floatsw_(i), float w_(io) Procedure: 1. Compute feature values from detectedpulses (n values per pulse, where n is dimensionality of feature space): feature Values = feature_extract(detectedPulseData) 2. Cluster featurevalues in feature space to obtain population memberships  populations =cluster (feature Values) 3. Compute 2^(nd) order statistics ofpopulations:  (for i = 1 to m, where m is number of populations/classes)  (for j = 1 to n, where n is dimensionality of feature   space)     ${{popMean}_{i}\lbrack j\rbrack} = \frac{{{sum}\left( {{featureValues}\left\lbrack {{populations}_{i},j} \right\rbrack} \right)} -}{\#\mspace{14mu}{of}\mspace{14mu}{samples}\mspace{14mu}{in}\mspace{14mu}{populations}_{i}}$   (for k = 1 to n, where n is dimensionality of feature    space)  tmpVal[j,k]= (feature Values[populations_(i), j] -    populationMean_(i), [j]) • (featureValues[populations_(i),        k] - populationMean_(i), [k])      ${{popCovariance}_{i}\left\lbrack {j,k} \right\rbrack} = \frac{{sum}\left( {{tmpVal}\left\lbrack {j,k} \right\rbrack} \right)}{\#\mspace{14mu}{of}\mspace{14mu}{samples}\mspace{14mu}{in}\mspace{14mu}{populations}_{i}}$4. Compute polynomial discriminant function coefficients:  (for i = 1 tom, where m is number of populations/classes)  W_(i)= -1/2 •popCovariance_(i) ⁻¹  w_(i) = popCovariance_(i) ⁻¹ • popMean_(i)        w_(io) = −1/2 • ln(|popCovariance_(i)|) −     1/2 • popMean_(i)^(T) • popCovariance_(i) ⁻¹ • popMean_(i) +    ln(popPriorProbabilities_(i))

In general, the A/D converter 689 converts the analog output signals 701from the photodetector 117 into corresponding digital information 707indicative of characteristic A or characteristic B (e.g., X or ˜X). Thedigital signal processor 867 generates initial discrimination parameterscorresponding to the digital information 707, discriminates the digitalinformation as a function of the initial discrimination parameters, andprovides a sorting signal 853 to the sorting system 119 as a function ofthe discriminated digital information.

G. Digital Pulse Detection

The first processing step is pulse detection performed by pulsedetection processor 865 to determine whether a particular waveform is awaveform pulse 497 corresponding to a fluorescence emission 31 of acell. The processor 865 executes a pulse detection algorithm whichidentifies sample sets that are likely to represent either particlestargeted for sorting into a population or particles targeted to beavoided because they are potential contaminants to a population. In thecase of bovine sperm sorting, a dye is added to quench the emission 31of non-viable cells, causing their associated pulse intensities to be ˜⅓the intensity of a live cell. Nonviable cells are not considered assorting targets or potential contamination. They are not considereddetected pulses 497. Pulses 497 from live cells are detected bymonitoring the intensity of samples for a successive number of samplesthat rise above the background levels. Once this level crosses astatistically determined threshold the processor 865 jumps to a latertime that is approximately 75% of the expected pulse 497 width for alive cell. If the level is still above the threshold, the series ofsamples are considered to be a pulse 497. Samples from detected pulses497 are moved to a block of memory used by the feature extractionprocessor 867.

A statistical anomaly detection approach is one embodiment which may beemployed by digital pulse detection software 747 although it iscontemplated that other approaches for identifying and/or isolatingdigitized pulses 497 may be used. Essentially, digital samples 707 ofthe output voltage signals 701 from the photodetector 117 detectingfluorescence which are statistically anomalous from the background areconsidered to part of a pulse 497. For additional robustness (tominimize noise detections), additional temporal criteria may beincluded.

Pulse detection proceeds as follows. When the voltage output signal 701from the photodetector 117 is not a pulse, the Mahalanobis distance fromthe background of incoming samples 707 of the signal 701 is computed andcompared with a preset threshold. If the distance of a given sampleexceeds the threshold, it is considered to be the potential start of apulse 497, and the pulse detection software begins to buffer theincoming samples. If the next predetermined number of samples (e.g., 25)also exceed the threshold, a pulse 497 is considered to have started andbuffering continues until the pulse end criteria are met; otherwise, thebuffer is reset and checking for the start of a pulse resumes. While ina pulse 497, if a sample is below the threshold, then it is consideredto be the potential end of a pulse and the buffer location is recorded(but sample buffering continues). If the next predetermined number ofsamples (e.g., 25) are also below threshold, the pulse 497 is consideredto have ended and the pulse 497 consists of the buffered samples up tothe recorded location. Table VI summarizes the pulse detectionalgorithm, and FIG. 49 provides an illustration of the results of pulsedetection on a digitally acquired fluorescence pulse 497.

TABLE VI Summary of digital fluorescence pulse detection. Algorithm:Digital fluorescence pulse detection Input: vector of floats digSamples,float bkgrndMean, float bkgrndSigma, float pulseStartThresh, floatpulseEndThresh, integer numStartSamples, integer numEndSamples Output:vector of floats pulseBuffer Procedure: 1. Initialize inPulseFlag = 0,pulseStartCount = 0, pulseEnd- Count = 0 2. For each sample indigSamples, compute Mahalanobis distance from background:${{mhDist}\lbrack i\rbrack} = \frac{\left( {{{digSample}\lbrack i\rbrack} - {bkgrndMean}} \right)}{bkgrndSigma}$3. If inPulseFlag is not set, go to step 4, else go to step 6. 4. IfmhDist> pulseStartThresh, place sample in pulseBuffer, incrementpulseStartCount, and go to step 5; else set pulseStartCount = 0, go tostep 2. 5. If pulseStartCount> numStartSamples, set inPulseFlag and goto step 2. 6. If mhDist < pulseEndThresh, place sample in pulseBuffer,set lastPulseSample to current buffer position, increment pulseEndCount,and go to step 7; else set pulseEndCount to zero and go to step 2. 7. IfpulseEndCount is greater than numEndSamples, return pulseBuffer[1 tolastPulseSample] and exit.

In general, the A/D converter 689 converts the analog output signals 701from the photodetector 117 into corresponding digital information 707indicative of characteristic A or characteristic B (e.g., X or ˜X). Thedigital signal processor 865 analyzes the digital information andprocessor 873 provides a sorting signal 853 to the sorting system 119 asa function of the detected digital information.

H. Feature Extraction and Discrimination

The next processing step is feature extraction performed by the featureextraction and discrimination processor 867. This processor responds toflags set by the pulse detection processor 865. Samples from detectedpulses are placed in memory shared with the feature extraction processor867. Features such as area, pulse width, pulse height, Gaussiancorrelation coefficient and/or other features are determined for eachpulse 497. In some cases pulses 497 are determined to be “doublets” orinvalid and features are not extracted. For the case of bovine sperm 201features are only extracted for pulses 497 that have the generalamplitude and width of a live X or Y cell. Typically, the pulseamplitude for a live sperm cell is in the range of about 700-900 mV,although this range may be as wide as 500-1000 mV. Once the features areextracted they are compared to the feature spaces defined for thepopulation(s) selected for sorting. If the features match the featurespaces identified for sorting, then processor 867 sets a flag indicatinga positive sort command to the sort processor 873. In general, theclassification of a particular cell is made by the discriminationprocessor 867 and the sort decision is made by the sort processor 873.

Digital information 707 representing fluorescence emissions 31 (and thusthe characteristics of corresponding cells which created them) arediscriminated by software 757 based on specific features orcharacteristics which exhibit distinguishably different statisticalbehavior in feature space (the n-dimensional orthogonal space formed byn features as the axes) for the different populations of interest.Therefore, the first step in analyzing digital information 707 for thepurposes of discrimination is computation of these features, a processcalled feature extraction performed by pulse analysis software 749executed by the processor 867. Table VII lists the several candidatefeatures which software 749 may use for this application. One or more ofthese features will be selected to form the feature space forclassification. It should be noted that there are additional featuresproviding enhanced separation so that this list is exemplary, notcomprehensive. For example, the software 749 may employ a subroutine 753to determine pulse 497 area and/or may employ a subroutine 755 todetermine pulse 497 peak.

TABLE VII Summary of candidate features currently being considered foruse in digital pulse analysis relating to feature extraction. FeatureName Feature Description Pulse Area Approximated by sum (or average) ofpulse samples Pulse Peak Maximum value of pulse samples Pulse “Inner”Area Sum (or average) of inner TBD samples of pulse (centered on pulsemean) Pulse Width Number of samples in pulse. Pulse “Gaussianity” MSE orcorrelation coefficient of pulse with a Gaussian shape with the same 2ndorder statistics. Pulse “Lagging Peak” Pulse value at TBD samples pastpeak (or mean) Critical Slope Slope of pulse at a point along the pulseat Difference (CSD) which the difference between the first derivative ofa pulse produced by particles having characteristic A and the firstderivative of a pulse produced by particles having characteristic B isat or near a maximum

I. Slit Scanning

In general, the elliptical spot 459 provided by the illumination system109 measures the relative DNA content differences in cells. Resolutioncan be improved further by analyzing the fraction of the pulse 497 ofthe fluorescence emission 31 detected by the photodetector 117 morelikely to contain characteristics which are being evaluated. Abiological phenomenon of certain cells (e.g., bovine sperm cells) is thelocalization of the X/Y chromosomes in a sub-equatorial region 225 whichis immediately adjacent the longitudinal midline or equator or center ofthe nucleus 213 of the cell 201 and which has a length of about 1 μm.(See FIG. 6). In fact, the X/Y chromosomes are not necessarily centeredin the nucleus 213. Thus, resolution can be improved by converting thetime-varying analog output 701 of the photodetector 117 into digitalinformation 707 and analyzing a portion of the digital informationcorresponding to the fraction of the pulse 497 of the fluorescenceemission 31, e.g., corresponding to the light emitted from thecircumequatorial region 225 such as 20-60% and particularly 20-30% ofthe waveform pulse centered around the pulse 497 peak.

As noted above, slit scanning can be employed to obtain the fluorescencemeasurement from a portion of each cell's chromatin rather than from thechromatin as a whole. The elliptical spot 459 provided by theepi-illumination system 415 noted above measures the relative DNAcontent differences in cells from specific sections of the chromatin, sothat the resolution of X cells and ˜X cells relative to one another isimproved. As noted above, the slit scanning measurement technique is afluorescence measurement approach that focuses the excitation beam 25 sothat a dimension of the focused spot size 459 is much less than a celldiameter as shown in FIG. 6. In this way, the cell 201 is scanned by thelaser beam 25 as the cell passes through the elliptically-shaped beamspot 459. The resulting waveform pulse 497 produced by the photodetector117 output 701 detecting the fluorescence emission 31 resulting fromslit scan illumination contains information about the localization offluorescence along the length of the cell 201. As shown in FIGS. 45-48,as the cell 201 traverses the elliptically-shaped beam spot 459, thetime-varying waveform pulses 497 (red/orange line) are the convolutionof the relative beam intensity (blue line) and the relative emittedpulse intensity (which corresponds to the fluorescence emissions fromstain excited by the elliptical spot as the cell traverses the beam andwhich varies because the fluorescence distribution along the axis of thecell varies).

By illuminating only a fraction of the cell's chromatin at one time, theresulting time-varying analog output 701 from the photodetector 117contains information specific to the localization of fluorescence withinthe chromatin along the longitudinal axis of the cell 201. Although thedetected fluorescence emission 31 from slit scanning is less than thedetected emission 31 from scanning by a beam 25 having a spot widthcomparable to the cell diameter, resulting in waveform pulses 497 fromslit scanning having a lower pulse amplitude, the majority of differencebetween the X-chromosome bearing cells and the Y-chromosome bearingcells appears in the center 20-30% to 20-60% of the waveform pulse 497.If only the rectangular area 725 in FIG. 53 is considered fordiscriminating X-Y sperm cells, then a larger relative difference can bemeasured between the localized variation in DNA content within thesection of chromatin that corresponds to the rectangular region 725 dueto the presence of the X and Y chromosomes within that region ascompared to the total DNA content of the cells. For example, bovine X-Ysperm cells have a difference in total DNA content of about 3.8%. Thefluorescence emission 31 from the X and Y chromosomes will be containedin the rectangular region 725. If this rectangular region 725 accountsfor 20% of the total waveform pulse 497 corresponding to a fluorescenceemission 31, then a 14% difference in relative DNA content within theregion will exist. By measuring the relative DNA content differencesfrom specific sections of the chromatin, the resolution of X-Y spermcell differentiation is improved (e.g., from 3.8% to 14%). FIG. 54illustrates the resolution attainable using slit scanning illuminationand processing the areas from only the center 20% of the pulse 497(i.e., the rectangular region 725 of FIG. 53). The histogram of FIG. 54allows a very high percentage (e.g., 98%) of the X chromosome bearingsperm and Y chromosome bearing sperm to be identified with a high degreeof confidence (e.g., 95%). In comparison, the histogram of FIG. 55,which illustrates the resolution obtainable when using standardillumination techniques, shows that slit scanning offers a significantimprovement over the results obtained using standard illuminationtechniques.

Two approaches which can be employed to obtain the area 725 of thecenter portion of the waveform pulse 497 as illustrated in FIG. 53 aredigital signal processing (DSP) of digitized photodetector 117time-varying analog output 701, as discussed in this section, or analogintegration using an analog threshold trigger, as noted below. As notedherein, DSP processing involves continuously sampling the time-varyinganalog output 701 from the photodetector 117 to obtain digitalinformation 707 corresponding to the output 701 and applying DSPalgorithms to the digital information 707 to extract features, such asarea size, from the digital information corresponding to the centerportion 725 of the waveform pulse 497 which corresponds to thedifference in DNA content due to the presence of an X or Y chromosome indifferent cells 201. As a simple example, the center 20% of the totalarea of each waveform pulse 497 would be determined by analyzing thedigital information 707 corresponding thereto. The analysis would beused to generate a histogram such as illustrated in FIG. 53.

J. Pulsed Laser Scanning

In one embodiment, it is contemplated that the system 1 include a pulsedlaser to illuminate the cells. In this embodiment, slit scanning (asdescribed above) may or may not be employed. For example, a mode-lockedsolid-state laser can be used to emit a train of electromagnetic pulseshaving a pulse width (duration) of 1-100 picoseconds at a pulsefrequency of about 50-150 MHz and at an average power output of about100-500 milliwatts. One suitable laser is a Vanguard 350 mode-lockedsolid-state laser (available from Spectra-Physics, Mountain View, Calif.94039), which is operable to emit a series of pulses about 12picoseconds in width (duration) at a frequency of about 85 millionpulses per second and at an average power of about 350 milliwatts.Because the 350 mW of power is delivered over extremely short bursts ofonly 12 picoseconds, the peak power output of such a laser is severalhundred times (e.g., about 800 times) greater than the average power.

The output of such a laser can be described as quasi continuous wave(quasi-cw) because, for many applications, the pulse repetition rate isfast enough to approximate a continuous wave (cw) output. Indeed it ispossible to operate the system as described above with a quasi-cw laserin much the same manner as one would operate with a cw laser. Thisprovides certain advantages because solid-state lasers typically operatemore efficiently, require less extensive cooling systems, and requireless maintenance than most other lasers.

A quasi-cw pulsed solid-state laser can also result in significantlyimproved signal-to-noise ratios using digital signal processingtechniques. A timing circuit may be included and is operable to producea timing signal indicative of the arrival of laser pulses at theinterrogation location 115 (i.e., the area where the laser beam 25illuminates the stream 21). For example, the timing circuit may be alaser pulse sensor 3003 as shown in FIG. 40 for detecting lightcorresponding to the laser pulse including scattered light generated bythe interaction of each laser pulse with the fluid stream 21 and/orincluding light from the laser pulses. Alternatively, for lasers whichmay be triggered, a triggering signal may be provided to themicroprocessor 131 and/or the A/D converter 689 to synchronize either orboth to the laser pulses, as noted below with regard to FIG. 50. Ineither embodiment, the laser pulse timing would provide a clock signalfor the system.

Referring to FIG. 50, a timing diagram illustrates the timingrelationship between the laser pulses LP, the fluorescence emissions FEfrom a cell as a result of repeated excitation by the laser pulses LP asthe cell passes through the beam spot 459 and the digital samples DS ofthe photodetector output 701. As shown in FIGS. 45-49, as a cell passesthrough the laser beam spot 459 the fluorescence emission 31 variesdepending upon the amount of illumination of the portion of the cellwhich generates the fluorescence emission 31. FIG. 50 illustrates twenty(20) laser pulses LP1-LP20 which impinge upon a cell as the cell passesthrough the interrogation zone 115 of a flow cytometer 1. Each laserpulse LP1-LP20 corresponds to a fluorescence emission FE1-FE20,respectively, which exponentially decays after substantiallyinstantaneous excitation by the laser pulse.

In one embodiment, the microprocessor 131 controls the A/D converter 689(see FIG. 40) so that the converter 689 samples the output signal 701 ofthe photodetector 117 at or near peak of each fluorescence emissionFE1-FE20, as indicated by digital samples DS1-DS20, respectively. Inother words, the timing circuit synchronizes the sampling rate of theA/D converter 689 with the fluorescence emissions FE1-FE20. Theresulting digital signal produced by transit of a particle through theinterrogation zone 115 is the functional equivalent of the digitalsignal that would have been produced by the digitization of a pulsewaveform 497 from a continuous wave laser. As shown in FIG. 51, forexample, by considering only the fluorescence intensity during thedigital samples DS1-DS20 and disregarding fluorescence intensitydrop-off between laser pulses LP1-LP20, the fluorescence intensity as afunction of time is a pulse waveform 497. This permits featureextraction by the microprocessor 131 from the digital signal 707generated by the pulsed laser in order to analyze the cell providing thefluorescence emissions FE1-FE20. In one embodiment, a more sensitivephotodetector having relatively fast response time of about 2nanoseconds or less may be used to more accurately detect thefluorescence emissions.

Thus, the pulsed laser provides advantages in a flow cytometry system 1in that it is possible to use a lower power pulsed laser to obtainsubstantially the same analysis that would be obtained with a cw laseroperating at an average power much higher than the average power of thepulsed laser. Further, the high peak power from a pulsed laser tends tosaturate the fluorophores so that the fluorescence emissions aremaximized thereby reducing the signal-to-noise ratio of the outputsignals of the photodetector. In other words, by using a laser pulsethat contains much more energy than is required to saturate thefluorophore, variations in the output of the laser do not result invariations in the fluorescent emissions 31.

Those skilled in the art will recognize that there are many ways tocause a laser to emit a series of pulses. It is understood that otherpulsed lasers, including other mode-locked lasers, Q-switched lasers,and cavity dumping lasers, could be used in place of the mode-lockedlaser discussed above without departing from the scope of thisinvention. Similarly, many other ways to time the digital sampling andprocess the resulting information will be apparent from the foregoingdisclosure. For example, the digital sampling could be timed so there isa different delay (or no delay) between a laser pulse and a digitalsample without departing from the scope of the invention. Likewise, thenumber of digital samples per pulse or the number of pulses that elapsebetween digital sampling can also be varied without departing from thescope of this invention.

K. Estimation of Population Characteristics

As noted above, flow cytometry can be used to discriminate X-bearingbovine sperm cells from Y-bearing bovine sperm cells based on theirrelative 3.8% difference in DNA content. Discrimination is achievedthrough analysis of characteristics of the time-varying signal 701 thatis produced by the photodetector 117 used to record the fluorescenceemission 31 as the stained cell passes through the interrogationlocation 115. This interaction is illustrated in FIGS. 45-48. FIGS.45-48 illustrate how a pulse waveform 497 is generated by thefluorescence emissions 31 resulting from the interaction between thelaser beam 25 and a stained sperm cell 201. The emission pulse 497 isthe convolution integral of the excitation spatial function 498 and theemission spatial function of the cell 201. Characteristics of thefluorescence pulse waveform 497 are used to classify a cell as X, Y orundetermined. In one embodiment, X-Y discrimination relies on two pulsecharacteristics: peak pulse height and pulse area.

These characteristics are illustrated on the example pulse that appearsin FIGS. 52 and 53. FIGS. 52 and 53 are examples of pulses 497 fromX-bearing and Y-bearing sperm cells. The pulses 497 were generated froma computer model that assumed excitation illumination with a laser beam25 having an elliptically-shaped beam spot 459 having a 2 μm Gaussianbeam waist W1 (FIG. 6) and that the DNA content difference wasdistributed uniformly across the center 20 percent of the cell 201.These assumptions are representative of slit scanning illumination ofbovine sperm cells 201 having a localized DNA difference as discussed inmore detail above. Integration of the pulses 497 results in a 3.8%average difference between the pulse 497 area for an X cell and thepulse 497 area for a Y cell.

It is possible to generate histogram and scatter plots of the pulse 497peak and area characteristics for stained cells and nuclei. FIGS. 56-59contain histograms of the pulse area characteristic for stained nucleiand live cells, plus scatter plots of the pulse 497 area and peakcharacteristics for stained nuclei and live cells. Some of the itemsthat may limit live cell discrimination, and ultimately the cell sortingrate are evident in these plots. Notably, the live cell histogram ofFIG. 57, and to a lesser extent the nuclei histogram of FIG. 56, have aleft shoulder that is typical of fluorescence intensity histograms formammalian sperm cells. It has been determined that the left shoulder isgenerated by one or more populations of slightly unaligned cells (i.e.,cells that generate relatively weaker fluorescence emissions due toslight deviations from the optimal alignment, but that are not so farout alignment to cause the relatively brighter fluorescence emission 31from the narrow edge 209 of the sperm head 205 to be collected by theoptics system 109). Only about half of the X cells can be easilyidentified in the live cell area histogram. The rest overlap with the Ycell population and the non-aligned cell populations. Even when peakpulse height is added, as shown in the scatter plots in FIGS. 56-59,X-bearing cell classification may be significantly limited.

FIGS. 60-61 illustrate the overlap of the X and Y populationdistributions. In FIGS. 60-61, a four-component computer model has beenapplied to raw data 6000 (FIG. 60) to estimate population statistics fortwo populations of non-aligned cells (6001, 6003), aligned live Y cells(6005) and aligned live X cells (6007) (FIG. 61). It is desirable todiscriminate the X and Y populations as a function of the coefficient ofvariation (CV) of the X and Y populations. For example, it is desirableto minimize the coefficient of variation (CV) of the X and Y populationsin order to improve discrimination. In particular, when a population ofsorted X cells is desired, it is desirable for the CV of the X cellpopulation to be less than 1.5%, more desirably about 1.3%, and evenmore desirably less than 1.3%. Traditionally, the CV of a distributionof fluorescence intensity of sperm cells has been considered withrespect to the distribution functions for only two populations (X andY). Quality control with respect to CV has been limited to crudesubjective estimation of the CV of the X and Y populations to decidewhether continued analysis or sorting is worthwhile.

According to one embodiment of the present invention, one function ofthe microprocessor 131 is to provide an automated estimation of the CVof the X population using the four-component model illustrated in FIGS.60-61. In order to estimate the CVs of the populations present in afeature (e.g. pulse area) distribution, it is necessary to estimate the2nd order statistics of the population distributions. This may beachieved by applying a model of a known form and finding the best fit ofthat model to the observed data.

Given the expectation of normally-distributed data, an approachconsisting of Parzen Window based non-parametric density estimation(utilizing a Gaussian kernel function) followed by application of aGaussian mixture parametric model has been chosen. Specifically, thefour-component model illustrated in FIGS. 60-61 consists of a sum (ormixture) of four uni-variate Gaussian distributions, with these fourcomponents being the feature distributions corresponding to aligned Xcells, aligned Y cells, and a two-component aligned cell population. Theparameters characterizing the model then are the population means(averages) (4), population standard deviations/variances (4), and priorprobabilities (expected % of the overall distribution) (4). These 12parameters can then be varied to achieve a best fit of the model to theobserved data histogram. With the model component parameters thusestimated, an estimate of the CV of a population of interest (inparticular, X cells) may be determined from the estimated populationstandard deviation and mean:

${CV} = {{\frac{{standard}\mspace{14mu}{deviation}}{mean} \cdot 100}\%}$

In order to reduce computational complexity, constraints have beenplaced on the model to reduce the dimensionality of the parameter space.In particular, the standard deviations of the model componentscorresponding to the aligned X and aligned Y populations have beenconstrained to be the same. Also, the aligned X and aligned Y componentshave been constrained to make up the same percentage of the overallmixture—thus the non-aligned populations are assumed 50% X cells and 50%Y cells.

Non-parametric density estimation is applied prior to model fitting toobtain an improved estimate of the total density function (being the sumof the component densities) underlying the raw histogram data. Thespecific technique applied is known as “Parzen Windows” (Duda, Hart, andStork, 2001), here utilizing a Gaussian kernel or window function due tothe assumed sum-of-Gaussian nature of the underlying density. Thestandard deviation of the Gaussian kernel is chosen to be 1% of thenumber of populated histogram bins; this value has been empiricallyobserved to provide adequate but not excessive smoothing of thehistogram. Each data point in the histogram then contributes a kernelfunction centered on the histogram bin containing the data point. Thedensity estimate is then obtained as the sum of the kernel functions.

The methodology chosen for variation of the model parameters to achievethe best fit to the data is known as Expectation Maximization (See DudaR. O., Hart, P. E., and Stork, D. G., 2001, Pattern Classification 2ndEd., John Wiley & Sons; and Moore, A., “Very Fast EM-based Mixture ModelClustering using Multiresolution kd-trees,” in M. Kearns and D. Cohn,Eds., Advances in Neural Information Processing Systems, pages543-549,1999, Morgan Kaufman).

The specific algorithmic implementation utilized is as follows:

-   -   1) Initial conditions for the model parameters are set. The top        two local maxima in the Parzen density estimate are used as the        initial Y and X mean locations (the initial amplitude of the        maxima for both X and Y populations being estimated as the        amplitude of the right peak). The initial X and Y population        variance is estimated as the variance of the data to the right        of the local minimum occurring between the left and right peaks        relative to the right peak location. Also, the initial X and Y        population prior probabilities are set as the percentage of all        points falling to the right of this local minimum. The initial X        and Y population Gaussian density estimates are then computed        using these parameters and subtracted from the total Parzen        density estimate. The mean and variance of the remaining data        points are then computed and used to initialize the        two-component aligned population model as follows. The two        population means are assumed (arbitrarily) to be 5% apart (2.5%        above and below the overall aligned mean). Given an (initial)        assumption of equal priors and equal variances, then, the        component variances are given by:

σ_(1,2) ²=σ_(tot) ²−¼(μ₂ ²−μ₁ ²)

-   -    where σ′ is the variance and μ is the mean of the respective        population.    -   2) Updated estimates of component population statistics (means,        standard deviations, and priors) are computed using the Parzen        density estimate. Each histogram bin location is weighted in the        statistical computations by the Parzen density estimate in that        bin. Additionally, each data point contributes to all component        population statistic computations weighted by the degree to        which that point is believed to belong to a given population,        based on the current component population parameters. This        degree of membership is determined as the ratio of a given        component population (Gaussian) probability density value to the        sum of all component population probability density values at        the data point. Thus we have (for all data points x in the        histogram, populations c_(p)∈{c_(x), c_(y), c_(u)}, and        population parameter vector θ_(p)[μ_(p), σ_(p), P_(p)])        population component memberships used in the computation of        updated parameter estimates given by:

$\mspace{20mu}{{P\left( {\left. c_{p} \middle| x \right.,\theta_{p}} \right)} = {{P_{p} \cdot \frac{1}{\sigma_{p}\sqrt{2\;\pi}}}{\exp\left\lbrack {- \frac{\left( {x - \mu_{p}} \right)^{2}}{2\;\sigma_{p}^{2}}} \right\rbrack}}}$${{membership}\left( {\left. c_{p} \middle| x \right.,\theta_{p}} \right)} = {\frac{P\left( {\left. c_{p} \middle| x \right.,\theta_{p}} \right)}{\sum_{n \neq p}{P\left( {\left. c_{n} \middle| x \right.,\theta_{n}} \right)}} \cdot {{ParzenDensityEstimate}(x)}}$

-   -    Updated means and variances are then computed using the Parzen        density estimate values weighted by these membership values,        with updated priors given by the average membership for each        component population over all data points.    -   3) Parameter updating procedure continues until all parameters        reach steady-state (i.e., stop changing significantly from one        update iteration to the next (or a maximum allowed number of        iterations occurs).

As previously mentioned the aligned X and Y populations are constrainedin this procedure to have the same variance and prior probability. Thisconstraint is achieved by using the average of the X and Y variance andprior values computed via the above procedure at each iteration.

Alternatively, a similar modeling approach can be applied to athree-component model (FIGS. 62-63) in which the cells comprising thetwo unaligned populations 6001, 6003 in the four-component model aretreated as a single Gaussian distribution 6011 rather than two distinctsubpopulations. The non-aligned cells can be modeled as third Gaussiandistribution (shown FIG. 63) having a mean and standard deviationdetermined by a best fit of the left shoulder and left major peak of theraw data 6010 (shown FIG. 62). The three-population model also hasestimated statistics for the aligned Y Population 6015 and aligned XPopulation 6017. One advantage of the three-component model is that itrequires only 9-dimensional parameter space (compared to 12-dimensionalparameter space required for the four-component model). However, it hasbeen found that the four-component model typically results in anestimated density function that more closely matches the raw data.

Those skilled in the art will recognize that a wide variety ofstatistical techniques can be used to estimate the characteristics ofthe aligned X and aligned Y populations. Thus, the four-component model,the three-component model, or other models may be implemented by anyparametric or non-parametric computer algorithms to estimate thecharacteristics of the aligned X cell and/or aligned Y cell populationswithout departing from the scope of this invention.

L. CV-Based Selection of Staining Conditions

Several factors affect the efficiency of sorting stained cells within apopulation into enriched subpopulations of cells. Among these factors isthe amount of differential fluorescence between the varioussubpopulations of cells within a stained population. Differentialfluorescence is affected by dye uptake, which varies based upon stainingfactors, such as for example, the concentration of the stain, the lengthof the staining period, the temperature at which staining occurs, andthe number and concentration of any additives that may be included withthe stain or added to the staining mixture. Accordingly, adjustments toany or all of these factors may be made to increase the sortingefficiency (the rate at which cells may be sorted into at least oneenriched subpopulation of cells with certain degree of purity and/or aminimal loss of desired cells) of the population of stained cells.Further, one can increase efficiency of a multi-sample sorting system byadjusting one or more of these factors for each sample, therebycountering any sample-to-sample variations. In the context of bovinesperm sorting, for example, sorting efficiency can be improved byadjusting one or more of the foregoing staining factors from one semensample to the next to counter bull-to-bull variations orsample-to-sample variations within the same bull.

A determination of the coefficient of variation (“CV”) for a givenfluorescence emission characteristic of a population of cells to besorted is one manner in which to determine if adjustments to thestaining conditions could be made to achieve a desired sortingefficiency. For example, one may adjust the staining conditions as afunction of the CV of any feature extracted from the pulse waveformgenerated by movement of a cell through the interrogation location, suchas any feature indicative of total fluorescence intensity or peakfluorescence intensity (including total fluorescence intensity and peakfluorescence intensity). As previously discussed in greater detail, CVis an indicator of the homogeneity or consistency of a distribution of ameasurable property or characteristic of a population, such as forexample a fluorescence emission characteristic of a particularsubpopulation of a given population. CV may be determined by dividingthe standard deviation of the measured characteristic of a sample by thesample mean. CV can also be determined automatically by the flowcytometry system 9, such as by implementation of the iterative CVestimation algorithm discussed in detail above. The lower the CV, thegreater the homogeneity or consistency of the distribution of themeasured characteristic.

As applied to the staining and separation of sperm cells, the CV of aparticular fluorescence emission characteristic for a sample of X and Ychromosome bearing sperm cells may be affected by the stainingconditions. The concentration of the stain, the length of the stainingperiod, the temperature of the staining mixture, and/or the number andconcentration of additives affect the CV of a given fluorescenceemission characteristic. Increasing the concentration of the stain, thelength of the staining period, and the temperature of the stainingmixture and/or decreasing the number and concentration of additives willgenerally lower the CV. Such conditions may be altered individually orin combination. In addition, if any one of these factors is such that itwould tend to increase the CV of a fluorescence emission characteristic,such as for example, by shortening the staining time, then any one ormore of the other conditions may be adjusted such that it counteractsthe effect of the first, such as for example, by increasing the dyeconcentration, with the overall result being a decrease in the CV of thefluorescence emission characteristic to a level sufficient to achieve adesired sorting efficiency.

Accordingly, by manipulating any one or any combination of these factorsin this manner, the CV of a fluorescence emission characteristic of theX and Y chromosome bearing populations may be decreased to a value thatenables sorting of the sperm sample into a subpopulation of genderenriched semen comprising a desired percent purity of X chromosomebearing cells.

Unfortunately, changes that tend to lower the CV of the X bearing spermmay have negative consequences such as increased cost or decreased spermmotility or fertility. For example, other things being equal it isdesirable to use lower stain concentrations and shorter staining periodsto minimize any harmful impact of the staining process on the sperm.With this in mind, one may predetermine a CV at which an acceptablesorting efficiency will be achieved. Thereafter, a fraction of the cellsample to be sorted is stained and subjected to flow cytometricanalysis. A fluorescence emission characteristic of the fraction isdetermined, and the fraction is classified into subpopulations basedupon the characteristic. The CV of the fluorescence characteristic isdetermined with respect to the cells of one of the subpopulations (anenriched subpopulation). If the CV of the fluorescence emissioncharacteristic of the cells of the enriched subpopulation is equal to orless than the predetermined CV at which sorting is to occur, then theremainder of the cell sample is stained according to the conditionsunder which the fraction was stained. The sample is thereafter sorted,for example, according to the methods disclosed herein. If the CV of theparticular fluorescence emission characteristic of the cells of theenriched subpopulation is greater than the predetermined CV at whichsorting is to occur, then another fraction of the same sample isanalyzed in a similar manner, but under staining conditions believed toachieve a yet lower CV. In such a situation, the CV may be lowered by,for example, increasing the length of the staining period, increasingthe concentration of the dye, increasing the temperature at which thefraction is stained, or any combination thereof. This series of steps(i.e., removal of a fraction from the sample to be sorted, adjustment ofthe staining conditions, and a determination of the CV) is repeateduntil the CV of the particular fluorescence emission characteristic ofthe cells of the enriched subpopulation is determined to be equal to orlesser than the predetermined CV. Thereafter, the remainder of thesample is stained accordingly and may be sorted, for example, accordingto the methods disclosed herein. In a particular embodiment of theinvention, the cell sample comprises a semen sample, and the cells ofthe enriched subpopulation comprise X chromosome bearing sperm cells.

Accordingly, one embodiment of the invention is a process for evaluatinga set of conditions for staining a population of cells for sorting, thepopulation comprising a first type and a second type of cell. Theprocess comprises (a) staining a fraction of the population of cellswith a fluorescent dye under a set of staining conditions; (b) exposingthe stained cells to electromagnetic radiation as the stained cells arepassed through an interrogation location of a flow cytometer at a rate,R; (c) determining a fluorescence emission characteristic of the exposedcells; (d) using the determined fluorescence characteristic to classifythe cells into two or more sub-populations, one of the subpopulationsbeing an enriched subpopulation of the first cell type; (e) determininga coefficient of variation for the fluorescence emission characteristicof the cells of the enriched subpopulation; and (f) determining whetherto modify any staining condition under which the cells are to be stainedor the rate, R, at which the stained cells are passed through theinterrogation location of the flow cytometer. In another embodiment,another fraction of the population of cells is stained under a differentset of staining conditions and steps (b) through (e) are repeated withthat fraction. This process may be performed on two, three, four or anynumber of additional fractions. In another embodiment, multiplefractions of cells are drawn from the sample at the same time. Eachfraction may be stained simultaneously, or each may be stainedsubsequent to the previous fraction being passed through the flowcytometer. In the former case, each fraction may be stained with its ownunique set of staining conditions and step (f) may comprise using therespective CVs to determine a set of staining conditions to be used tostain additional cells. In the later instance, the staining conditionsof the subsequently stained fractions may be altered according to thedetermination of step (f) with respect to a previously analyzedfraction. In one embodiment the process is repeated until the CV isdetermined to be about equal to or less than a specified CV (e.g.,1.3%).

Alternatively, once one has predetermined a CV at which an acceptablesorting efficiency will be achieved, the entire cell sample may bestained. A fraction of the cell sample is removed and subjected to flowcytometry analysis. A fluorescence emission characteristic of thefraction is determined and used to classify the cells into two or moresub-populations. The CV of the fluorescence characteristic is determinedwith respect to the cells of an enriched subpopulation. If the CV of thefluorescence emission characteristic of the cells of the enrichedsubpopulation is equal to or less than the predetermined CV at whichsorting is to occur, then the remainder of the cell sample is thereaftersorted. If the CV of the particular fluorescence emission characteristicof the cells of the enriched subpopulation is greater than thepredetermined CV at which sorting is to occur, then a second fractionfrom the same sample is analyzed in a similar manner and the CV of thesame fluorescence characteristic is determined. The CV of the secondfraction may be lowered by, for example, increasing the length of thestaining period, increasing the concentration of the dye, or anycombination thereof. This series of steps (i.e., removal of a fractionfrom the sample to be sorted and a determination of the CV) is repeateduntil the CV of the particular fluorescence emission characteristic ofthe cells of the enriched subpopulation is determined to be equal to orless than the predetermined CV. Thereafter, the remainer of the samplemay be sorted, for example, according to the methods disclosed herein.In a particular embodiment of the invention, the cell sample comprises asemen sample, and the cells of the enriched subpopulation comprise Xchromosome bearing cells.

Accordingly, another embodiment of the invention is a process forevaluating a set of conditions for staining a population of cells forsorting, the population comprising a first type and a second type ofcell. The process comprises (a) staining a fraction of the population ofcells with a fluorescent dye under a set of staining conditions; (b)exposing the stained cells to electromagnetic radiation as the stainedcells are passed through an interrogation location of a flow cytometerat a rate, R; (c) determining a fluorescence emission characteristic ofthe exposed cells; (d) using the determined fluorescence emissioncharacteristic to classify the cells into two or more subpopulations,one of the subpopulations being an enriched subpopulation of the firstcell type; (e) determining a coefficient of variation for thefluorescence emission characteristic of the cells of the enrichedsubpopulation; (f) determining whether to modify any staining conditionunder which the fraction of cells are to be stained or the rate, R, atwhich the stained cells are passed through the interrogation location ofthe flow cytometer; and (g) applying the modified staining condition tothe remainder of the population of cells. In another embodiment, steps(a) through (f) are repeated at least once with at least one otherfraction of the population of cells. Steps (a) through (f) may berepeated once, twice, three times, four times or a greater number oftimes. In another embodiment, multiple fractions of cells are drawn fromthe sample at the same time. Each sample may be stained simultaneously,or each may be stained subsequent to the previous fraction being passedthrough the flow cytometer. In the later instance, the subsequentstaining of the fractions may be altered according to the determinationof step (f) with respect to a previously analyzed. In still anotherembodiment, the process further comprises prior to step (g), selectingthe modified staining condition that results in the lowest coefficientof variation for the fluorescence emission characteristic. In yetanother embodiment, the process comprises the repetition steps (a)through (e) until the coefficient of variation for the fluorescenceemission characteristic of at least one of the fractions is about 1.3%or less. In another embodiment of the invention, the process furthercomprises prior to step (g), selecting the modified staining conditionthat results in a coefficient of variation of about 1.3 or less.

In addition to performing such an analysis before sorting the entiresample as detailed above, a similar analysis may be performed while thestaining and sorting of the sample is occurring in an effort to ensurethat sorting efficiency is maintained. Accordingly, in anotherembodiment, the CV of a fluorescence emission characteristic of thecells of an enriched subpopulation of a fraction of a sample that hasbeen previously stained, a portion of said sample which is in theprocess of being sorted, is determined as described above. Adjustmentsto the staining conditions under which these samples were stained aremade according to the methods discussed above with respect to thepresort adjustments.

The selection of a predetermined CV at which an acceptable sortingefficiency will be achieved is based upon several factors, including forexample, the type of cell being sorted, the rate of sorting, and thedegree of purity desired with respect to sorting of the population intoenriched subpopulations. Generally, a CV is selected that will allow forsorting to the desired percent purity of the enriched subpopulationwhile minimizing the amount of time necessary to achieve the same, suchas for example, by achieving an 85% degree purity of the enrichedsubpopulation while minimizing the length of the staining period. Withthese factors in mind, the CV of the fluorescence emissioncharacteristic of the cells of an enriched subpopulation is generallybetween about 2.0% and about 1.0%, preferably between about 1.5% andabout 1.0%, more preferably about 1.4%, and still more preferably about1.3%.

M. Critical Slope Difference Feature Extraction

The microprocessor 131′ with digital signal processing (DSP) illustratedin FIG. 40 employed as part of a cell sorter makes it possible toextract features of the time resolved fluorescence emission pulse,particularly features that cannot be easily or inexpensively obtainedusing analog technology. In particular, a pulse feature which exhibitsnon-linear properties and which significantly improves the separationand thus the resolution of particles A and B (e.g., improves thediscrimination of live, aligned X sperm cells) is a feature referred toas critical slope difference (CSD). CSD is a quantification of the slopeof the fluorescence emission pulse at a signal amplitude where thedifference between the first derivative of a pulse produced by particleA (e.g., an X-bearing cell) and the first derivative of a pulse producedby particle B (e.g., a Y-bearing cell) approaches a maximum.

Functions that describe fluorescence emission pulses may be expressed interms of signal amplitude as a function of time: y=x (t). Within thecontext of detecting CSD features, a function may be defined thatdescribes the fluorescence emission pulses in terms of pulse durationtime as a function of signal amplitude. This function may be referred toas an M function. The M function is obtained by transposing thefluorescence emission pulse function as shown below.

Fluorescence Emission Pulse function: y=x (t)

M Function: t=M(y)

t=pulse duration

y=signal amplitude

Comparison of the M functions for typical X and Y bovine sperm cellsillustrates the discriminating power of the CSD feature. The top panelof FIG. 64 shows average M plots for X-bearing and Y-bearing spermcells. The middle panel in FIG. 64 shows a graph of the firstderivatives of these average M plots (i.e. M′) for signal amplitudevalues less than the peak height of the average Y-bearing fluorescenceemission pulse. It can be seen in this plot that as signal amplitudeapproaches the average peak height of the Y-bearing pulse, thedifference between the first derivatives (M′y and M′x) increasessignificantly. Plotted in the bottom panel of FIG. 64 is the differencebetween the first derivatives (M′x-M′y) as a function of signalamplitude. The CSD feature quantifies the slope of M (M′) for anindividual pulse near the region where the maximum difference in firstderivatives occurs (or the slope at a corresponding point on thefluorescence emission pulse function). For the purpose of discriminatingX and Y bearing sperm cells, CSD is determined for each pulse at thepoint where the leading edge of the pulse intersects the CSD threshold,as shown in FIGS. 62-63. In some embodiments, CSD may depend upon thecharacteristics of the illuminating beam such as beam width whether thebeam is continuous or pulsed. An algorithm for determining CSD isdiscussed below with regard to FIG. 65.

FIG. 64 illustrates that in some cases the CSD feature has a non-linearnature, such as in the case of sorting X-Y sperm cell populations. Thedifference between the derivatives (M′x-M′y) increases as the CSDthreshold approaches the peak of the Y pulse. The nonlinearcharacteristic of this difference places the mean value of thenonaligned cells and the aligned Y cells 45% lower than the mean valueof the aligned X cells in the CSD feature space. The standard deviationin the CSD feature space of the aligned X cells is largely unaffected(i.e. similar to that seen in the peak or area feature spaces). It isthis nonlinear, high gain nature of the CSD feature that increases thenumber of aligned X cells that can be accurately discriminated.

One computational efficient method for determining the CSD value for agiven pulse is illustrated in FIG. 65. A CSD threshold may be determinedas a function of a peak height of the fluorescence emission pulses. Inparticular, it may be determined based on the average peak height of thefluorescence emission pulses. The CSD threshold is maintained at a pointwhere about 25% of the pulse peaks from live, aligned cells fall at orbelow the threshold. Therefore, the CSD threshold is adjusteddynamically during the sort based on a running peak height distribution(i.e., relative to an average peak height). For example, the thresholdmay be based on a weighted running average of peak height (with morerecent measurements being given more weight than older measurements).The CSD value is the slope of a line that passes through two points onthe pulse. These points are the CSD pulse threshold and the pulse peak.Thus, in this embodiment the CSD value is only an approximation of theslope of the pulse waveform 497 at the intersection of the leading edgeof the pulse and the CSD threshold. However, other methods of computingthe CSD value for a given pulse are readily apparent, some of which canprovide more precise CSD values if desired.

In another embodiment, the CSD threshold is dynamically adjusted as afunction of the CV of the CSD feature extraction for a subpopulation ofparticles. In the case of sorting sperm cells for example, by increasingthe CSD threshold from a relatively low level (e.g., the pulse detectionthreshold) the CSD threshold will reach a level that results in asubstantial increase in the CV of the CSD of the Y cells but is stilllow enough that the increase in the CV of the CSD for the X cells issignificantly lower in comparison to the CV increase in the Y cells.This effect can be observed in the CSD distribution as a fanning out ofone subpopulation in the overall CSD distribution. Good discriminationfrom the CSD feature can be achieved by maintaining the CSD threshold atthis level.

It should be noted that the discriminating power of the CSD feature isenhanced by use of slit scanning approach to flow cytometry. The shapeof the beam spot 459 can influence the shape of the pulse waveform 497.For example, by using a beam spot 459 having a relatively small widthW1, a localized fluorescence difference in a sample particle (e.g., thelocalized fluorescent intensity difference resulting from localizationof the X or Y chromosome in the central region 225 of a sperm nucleus213) has a greater influence on the first order derivative of the pulsewaveform. Accordingly, one embodiment of the present invention includesusing the slit scanning techniques in combination with CSD featureextraction. Conversely, using a laser having a beam waist that is toolarge (e.g., equal to or greater than the diameter of the particles) mayprevent effective use of the CSD feature to discriminate particles. Theacceptable range for the width of the beam waist of the focusedillumination beam will depend on a number of factors including the sizeand shape of the particles, the distribution of dye within the particlesbeing analyzed, and the amount of difference between the typicalwaveform pulses for the particles to be discriminated. In the case ofsperm cells, CSD feature extraction from waveform pulses 497 generatedby excitation of bovine sperm cells 201 with a laser having a beam waistof less than 3 μm has worked well as indicated below. Of course, CSDfeature extraction with any form of slit scanning discussed in the slitscanning section is considered to be within the scope of this aspect ofinvention.

Use of the CSD feature substantially increases the yield of the system,particularly in the case of sorting X-Y sperm cell populations becauseit allows collection of many more aligned X cells. Due to the overlap inthe populations defined in peak vs. area or rise-time vs. area featurespaces, no more than about 70% of the aligned X cells can bediscriminated with a certainty about or greater than 85%. When the CSDfeature is used, 95% or more of aligned X cells can be discriminated,which significantly increases the percentage of live X cells that can becollected without reducing the purity of the population of collected Xcells below a desired level of purity.

This is seen graphically in the live cell data shown in FIGS. 66-69. Thenon-linear nature of the CSD feature allows X cells to be isolated forsorting. The gross selection on CSD applied in the scatter plot shownFIG. 68 results in a 70% pure X area population. When bi-variate sortdiscrimination is applied in the area and CSD feature spaces (FIG.68), >95% of the aligned X cells can be discriminated for sorting. Thedata in FIGS. 66-69 were collected at a total cell throughput of about22,000 live cells per second on one channel of a four-channel cytometrysystem (see multi-channel system discussion below). Even withcoincidence detection enabled (high purity), over 6,000 X cells persecond were collected at a purity level of at least 85% purity.

FIGS. 66-69 illustrate one advantage of the CSD feature when used todiscriminate X-bearing and Y-bearing sperm cells. FIGS. 66 and 67 arehistograms of the area feature of the fluorescence emission pulses forthe feature space defined in the scatter plots shown FIGS. 68 and 69. InFIG. 68, the CSD feature has moved most of the non-aligned and aligned Ycells completely out of the display of the scatter plot. This leaves a70% pure X population in the \ frame of the scatter plot, which is whatis shown in the pulse area histogram in FIG. 66. Non-CSD discriminationis shown in the pulse area/rise time scatter plot shown in FIG. 69.Aligned X cells make up about 30% of the corresponding area histogram(FIG. 67). More than 95% of the aligned X cells can be collected at >85%purity using the CSD feature for discrimination. By comparison, no morethan 70% of the aligned X cells can be discriminated using thetraditional feature space on the right.

Several live cell sorts have been completed using the CSD vs. pulsearea, bi-variate discrimination technique. FIG. 70 is an example of howa sort region set in this two dimensional feature space can be used toexclude non-aligned cells and Y cells. FIG. 70 illustrates a bi-variatesort region set on a scatter plot of CSD vs. pulse area scatter. Noticehow the sort region drops lower on the area feature for high values ofCSD (CSD increase from left to right and area increases from bottom totop) and moves higher on the area feature as CSD drops to lower values.The bi-variate sort region set on the above CSD vs. pulse area scatterplot was used to sort X cells at a sort decision rate >6000 X cells persecond with an input live cell rate of 26,000 cells per second. Puritybased on flow cytometry re-analysis was 87%.

The CSD feature makes possible a high yield, no-coincidence abort (i.e.,coincident accept or high recovery) sorting strategy. In someembodiments, a pulse feature could provide nearly baseline separationand thus 100% accurate classification of live X and Y sperm cells. Thiscondition would make it possible to sort cells at reasonably high rateswithout aborting droplets that contain both a cell classified as X andnon-X (either unknown or Y). This sorting strategy is referred to as thehigh recovery or coincidence accept strategy. An experiment wasperformed to test this using the CSD feature. Coincidence accept sortswere performed with an input rate of 12,000 live X cells per second onone channel of a four-channel flow cytometer. 77% of the X cells wereproperly aligned, making 4,600 X cells per second potentially availablefor sorting. Under these conditions, 4,300 cells per second were sortedinto the population of X cells. Subsequent purity analysis indicated apurity from this sort of >87% without correction for dead cells and 89%with correction for dead cells. A high purity, coincidence rejectdetection sort was performed immediately after this sort under the sameconditions. A collection rate of 3200-3500 cells per second wasobserved. Purity analysis indicated a purity of 92% without correctionfor dead cells and a purity of 94% with dead cell correction.

The results of the above experiment indicate that at 12,000 live cellsper second input, >92% of aligned X cells can be collected at apurity >85%. This is an indication that the CSD feature provides 95%accurate classification of all aligned X cells. Under thesecircumstances, yield from the cell sorter is limited primarily bycorrect cell alignment.

FIG. 71 illustrates one embodiment of flow cytometry re-analyses for atest in which the left panel corresponds to the high recovery coincidentaccept sort strategy (no coincidence abort strategy) and the right panelcorresponds to the high purity/coincident reject sort strategy(coincident abort strategy). The left panel (87% pure) was for an outputof 4,400 X cells per second without coincidence aborts. The right panelwas from a sort completed under the same conditions except dropletscontaining contaminating cells were aborted. Purity for this sort wasabout 92%. These sorts demonstrate that high recovery, no coincidenceabort sorts are possible when the CSD feature is used fordiscrimination.

Use of the CSD feature is not limited to sorting of sperm cells or anyparticular species of sperm cells. As those skilled in the art willappreciate from the foregoing disclosure, the CSD feature can be adaptedto improve discrimination between any groups of particles that generatesignal pulses having different first order derivative characteristicsregardless of the cause of the difference.

N. Discrimination

Once the features of the pulses have been extracted by pulse analysissoftware 749, discrimination (e.g., classification) of pulses isaccomplished by pulse discrimination software 757 executed by processor867 employing a logic application such as Bayes Minimum Risk decisionrule. This rule is a modification of a Bayes Minimum Error decision rulethat allows assignment (and adjustment for) differing costs associatedwith making different erroneous classification (e.g., discrimination)decisions.

Bayes Minimum Error computes the decision boundary 763 or decisionsurface as the surface of equal a posteriori probability betweenpopulations in feature space. For the case of (assumed) Gaussianprobability distributions this surface is in general quadratic, althoughin certain conditions may be linear (or be able to be closelyapproximated by a hyper-plane). The classification (e.g.,discrimination) decision is made by first computing the a posterioriprobabilities for a given point in feature space (generally fromclass-conditional probability densities and known/assumed a prioripopulation probabilities using Bayes Rule) then choosing the class labelas that of the population having the highest a posteriori probability.

Bayes Minimum Risk includes a factor to allow adjustment of the decisionboundary 763 in the case when it is desired to assign different costsfor making different classification errors (e.g. it may be costlier toclassify “Y” cells as “X” cells than vice versa). In this application,this allows a trade-off between sorted sample purity and recovery. Inthis decision rule, the “risk” of assigning each possible class label toa point in feature space is computed as the sum of the a posterioriprobabilities of membership in each population times the cost associatedwith classifying as the current population given true membership in eachother population. Table VIII summarizes the procedure for Bayes MinimumError classification. Note that for multi-variate Gaussian densities,evaluation of Bayes rule to obtain the aposteriori probabilities may bereduced to evaluation of the quadratic function seen in Table VIII,given that the coefficients W, w, and wo are as computed in thediscrimination algorithm parameter initialization procedure given inTable V. FIG. 74 shows a graphical example of classification by thisprocedure. The illustration on the left is a schematic illustration ofthe two populations 1 and 2 and the decision boundary 763 separating thepopulations. The histogram on the right shows two concentric sets ofellipses defining the X and Y regions, with the decision boundary 763being a line defined by the intersection of the ellipses.

TABLE VIII Summary of digital fluorescence pulse classification(discrimination) by Bayes Minimum Error decision rule. Algorithm: BayesMinimum Error fluorescence pulse classification (discrimination) Input:vector of floats pulseFeatures, for each class population i: matrix offloats W_(i)vector of floats w_(i), float w_(i0) Output: integerclassLabel Procedure: 1. For each class/population i, compute value ofdiscriminant function g_(i): g_(i) = pulseFeatures^(t) · W_(i) ·pulseFeatures + w_(i) ^(t) · pulseFeatures + w_(i0) 2. For eachclass/population i, compute value of risk function risk_(i): Initializerisk_(i)=0, then for each class/population j: risk_(i)=risk_(i) +cost_(ij) * g _(j) 3. Find j s.t. risk= min(risk_(i)) ∀i. ReturnclassLabel = j and exit.

For additional robustness, an additional step is taken in theclassification of digital fluorescence pulses. The Mahalanobis distanceof a pulse in feature space from the population assigned via BayesMinimum Error is computed, and if greater than a threshold, the pulse islabeled as “not classified” or some other appropriate indication that itis not likely a member of any known population. FIG. 75 illustrates theeffect of this additional step, again using features computed fromdigitally acquired fluorescence pulse data.

In general, the A/D converter 689 converts the analog output signals 701from the photodetector 117 into corresponding digital information 707indicative of characteristic A or characteristic B (e.g., X or ˜X). Thedigital signal processor 865 extracts features from the digitalinformation and processor 873 provides a sorting signal 853 to thesorting system as a function of the extracted features.

O. Sort Classification and Droplet Synchronization

The fourth, sort processor 873 manages droplet classification,implements sorting strategy and delivers a sort trigger pulse 853 thatis synchronized with the droplet selected for sorting. This processor873 receives cell classification information from the discriminationprocessor 867 and relates that information to the droplet generationclock 703 (i.e. aligns the position of particles classified for sortinginto a population with the formation of droplets). It determines ifthere is coincidence within a droplet and manages that coincidence basedon pre-determined sort strategies. It maintains a FIFO of all cellclassifications and droplet sort decisions that sets the correct delaybetween when the particle was observed in real time and when theparticle arrives at the last attached droplet. It will produce aproperly timed output pulse 853 of appropriate polarity and amplitudefor each droplet selected for sorting.

In general, the A/D converter 689 converts the analog output signals 701from the photodetector 117 into corresponding digital information 707indicative of characteristic A or characteristic B (e.g., X or ˜X). Thedigital signal processor 867 discriminates the digital information 707as indicative of characteristic A or as indicative of characteristic B(e.g., X or ˜X) and provides a sorting signal 853 to the sorting system119 as a function of the discriminated digital information.

In general, the digital signal processors 863, 865, 867, 873 includeinstructions for detecting waveform pulses represented by the digitalinformation, instructions for extracting features in the detected pulsesand instructions for discriminating the detected pulses as a function oftheir extracted features. In addition, the processors includeinstructions for defining a decision boundary 763 discriminating betweenthe extracted features representing characteristics A and the extractedfeatures representing characteristic B. Further, the processors 863,865, 867, 873 may optionally adjust the relative location of thedecision boundary 763 with respect to the extracted featuresrepresenting characteristic A and with respect to the extracted featuresrepresenting characteristic B as a function of at least one of thefollowing: (1) the purity of the at least one population with respect toeither characteristic A particles or characteristic B particles, and (2)the quantity of characteristic A particles or characteristic B particlesin the at least one population relative to the total quantity ofcharacteristic A particles or characteristic B particles in the stream.For example, the processor may move the decision boundary 763 to includeless of population 1 and more of population 2, or visa versa, based onthe output of a particular sample or based on the desired output (e.g.,as noted above with respect to the Bayes Minimum Risk decision rule toadjust the decision boundary for differing costs).

P. Drift Compensation

Given that over time the waveform pulses corresponding to thefluorescence emissions may vary or exhibit drift over time (due tostaining variations, temperature change, sample age and/or otherfactors, for example), the system may optionally employ drift analysissoftware 761 (FIG. 72) defining dynamic thresholds which vary tocompensate for any effects of drift. In particular, the pulse detectionthresholds employed by software 747 may be adjusted for any slowvariations in the signal background characteristics, and thediscrimination algorithm of software 757 may adjust the decisionboundary 763 (FIG. 74) to account for any drift in the populations infeature space.

In the case of the algorithm(s) employed by pulse detection software747, the drift compensation software 761 accomplishes drift compensationby updating the background mean and standard deviation estimates basedon sample statistics estimates computed within a moving window of agiven length of samples (e.g., 10-100 samples) ending with the currentsample. Given the (assumed) slow drift rate relative to the dataacquisition frequency, the background statistics need not be updatedevery sample; rather, background statistic updates may occurperiodically (e.g., every 6 seconds; see reference character 795 andFIG. 82). Additionally, the window may contain less than unity weightingto allow a “forgetting” rate to de-weight older samples relative tonewer samples in the statistics computations. FIG. 76 illustrates theconcept of statistic (mean) computation within a moving window withoutand with a “forgetting” rate.

Similar to the detection algorithm drift compensation, thediscrimination algorithm(s) employed by pulse discrimination software757 achieve drift compensation by periodic updates of the 2^(nd) orderstatistics of the populations in feature space. In this case, however,only those feature values from pulses assigned to a given population areused to update the statistics of that population. Again, non-unityweighting may be used to include a “forgetting” rate. FIG. 77 shows aconceptual illustration of the effects of applying this technique topopulations in feature space. FIG. 77 illustrates an example of driftcompensation for population statistics in feature space. Yellow denotespopulation 1 (X), green population 2 (Y), diamonds the class meanestimates (with an exaggerated illustration of drift), and block arrowschanges in the population covariance estimates reflected in deformationof the constant-sigma ellipses.

In general, the digital signal processor 863 employs a detectionthreshold for analyzing the digital information, which threshold is afunction of a background mean estimate and a standard deviation of thesampled time-varying output signals computed within a moving window ofsamples ending with the current sample.

Q. Advantage of all Digital Techniques Over Analog Techniques

One of the main advantages for using an all digital system for sortingis that there is no “dead time” associated with the detection andanalysis of a pulse. With analog systems there is always a finite“switching time” required for electronics to reset after the occurrenceand detection of a pulse. This time is usually on the order of at leastone microsecond. Since the digital system captures a continuous streamit really has no dead time.

Another advantage of a digital system is the ability to look forward andbackward in time around a pulse classified for sorting. In general, thedigital signal processing requires about five (5) droplet periods foranalysis. Preferably, the time delay between droplet illumination 115and droplet formation 107 is about seven (7) droplet periods. Thisallows the system to classify a particular particle based on theprobability that it will contaminate the usable population as indicatedby the features of the particular particle and based on the proximity ofthe particular particle to another classified particle. As an example,the sort processor 873 may reject a particle viewed as having a 50%probability of being a live X cell whereas the sort processor 873 mayaccept a particle viewed as having a 50% probability of being a live Xcell when the particle is coincident with a second particle viewed ashaving a 95% probability of being a live X cell.

R. Analog Cell Analysis

It is also contemplated that the time-varying output signals from thephotodetector may be processed by analog circuitry 819, such as a fieldprogrammable gate array, which may be less expensive than a digital cellanalyzer. FIG. 42 is a block diagram of one embodiment of an analog cellanalyzer which may be employed as part of the system according to theinvention. FIG. 53 graphically illustrates the analog analysis. Athreshold is set to produce a trigger based on pulse height. Thethreshold opens an integration window which gates an analog integratorto accumulate charge. The window remains open either for a fixed periodor until the pulse amplitude fall below the trigger threshold. In thisway, only the area of the portion of the pulse within the integrationwindow is used for relative fluorescence measurements.

Referring to FIG. 42, the output 701 of the photodetector 117 issupplied to an integrator 825 which integrates the output signal 701 insynchronization with the droplet clock 703. The integrated signal isprovided to a width/area comparator 827 for comparing the level of theintegrated signal to a threshold level defining a pulse (e.g., a pulsemay be defined as an integrated signal with 40% of a certain threshold).A dynamic threshold calculator 829 functions similarly to the driftcompensation noted above in that monitors the integrated signal leveland it varies the threshold level which the width/area comparator usesas a function of variations in the average integrated signal level.

The pulse discriminated signal is provided to a window comparator 837 toconfirm that the pulse area is within an acceptable range. The pulsediscriminated signal is also provided to a pulse width and trigger logiccircuit 839 to confirm that the pulse width is within an acceptablerange. If the area and width are acceptable, the logic provides atrigger signal to an I/O controller 843 which indicates the sortdecision 841. Thus, the window comparator 837 and the pulse width andtrigger logic 839 make the decision as to whether a cell should beclassified as an X cell or a ˜X cell.

The I/O controller 843 provides the sort decision 841 to the sortcontroller board 847 in the form of an X or ˜X signal. The I/Ocontroller 843 also includes a Universal Serial Bus (USB) interface 849for connecting to the PC 735 and may have I/O port for connecting toslave controllers 845 of the other channels. The analog cell analyzeralso includes a Joint Test Access Group (JTAG) port 833 for programmingthe width/area, comparator, the window comparator and the pulse widthand trigger logic.

It is also contemplated that the analog cell analyzer may be employedsimultaneously with the digital cell analyzer 705. For example, theanalog analyzer may be used to adjust voltage thresholds used by thedigital analyzer. On the other hand, the digital analyzer may be used toidentify various features of the pulse and this feature information maybe used to configure the analog cell analyzer, particularly if it isimplemented with a gate array.

Control Strategies

In general, the microprocessor 131 is programmed to implement controland sorting strategies which are intended to optimize the efficiency ofthe system 1 in terms of throughput and/or loss of desirable particlesto meet any cost requirements of the sorted product. This may involve,for example, balancing the need for high purity of at least onecollected population and the need to recover at least a minimumpercentage of desirable particles from the sample being sorted.Achieving such a balance is important, particularly in the context ofcommercial applications where cost and profitability are importantconsiderations.

To this end, the microprocessor 131 implements a control strategy whichis a series of instructions and/or algorithms that control systemvariables such as fluid delivery rate and/or sort parameters. Themicroprocessor also implements a sorting strategy which defines thedecision process for determining how each particle or group of particlesis sorted. Each particular control strategy may employ one or more sortstrategies. Various sorting strategies may be used depending on suchfactors as the selected control strategy, the particle detection systemand/or information relating to the particle distribution in the fluidstream.

Regarding particle distribution, FIG. 78 illustrates a fluid streamcontaining an exemplary distribution of particles. In this particularexample, the stream is formed by a nozzle similar to the nozzledescribed above and contains a mixture of particles having differentcharacteristics A and B, e.g., X and Y sperm cells. As shown, the cellsfollow generally one after another in a series which can be viewed ascomprising sequential sets of particles. These sets include firstparticle sets each comprising one or more particles having acharacteristic A (e.g., indicating a live X sperm cell), second particlesets each comprising one or more particles having a characteristic B(e.g., indicating a Y sperm cell or, more generally, a sperm cell whichis not a live X cell (˜X), such as a Y cell, or a dead X or Y cell), andthird particle sets each comprising two or more closely spaced particlesat least one of which has characteristic A and at least one of which hascharacteristic B (e.g., one more X sperm cells and one or more-X spermcells). Third particle sets are also hereinafter referred to as“coincident” particle sets.

Whether a particular particle is considered as constituting a set byitself or part of another set will depend primarily on its spatialposition and/or separation relative to adjacent particles. For example,in a droplet sorting system, the various particle sets will be definedby the particles in the droplets. In a photo-damage sorting system wherea laser is used to ablate (kill or otherwise damage) selected particlesets to provide a collected population having a desired content, asdiscussed below in the “Photo-Damage Sorting” section, the variousparticle sets will be defined by the spatial proximity of the particles,i.e., whether the spatial separation between particles is sufficient toenable accurate classification of the particles and/or the ablation ofone or more undesired particles by the laser without also ablating oneor more desired particles. Similarly, in a fluid-switching sortingsystem where portions of the stream containing selected particles arediverted to provide a collected population having a desired content, asis discussed below in the “Fluid Switching Sorting” system, the variousparticle sets will be defined by the spatial proximity of the particles,i.e., whether the spatial separation between particles is sufficient toenable accurate classification of the particles and/or diversion ofselected particles.

It will be observed from the foregoing that sort decision applied to thedifferent particle sets may be varied, depending on the desired resultor throughput of the system. For example, in a droplet sorting system,the sorting strategy used may depend on the treatment of “coincident”droplets, i.e., droplets containing third particle sets. In the handlingof bovine sperm cells in a flow cytometry droplet sorting system andmethod as described herein, for example, to enhance the number of Xsperm cells in at least one collected population, it may be desirable touse a strategy where each coincident droplet containing an X sperm cellis accepted and sorted as if it contained only X sperm cells, eventhough the droplet may also contain an ˜X sperm cell (coincident acceptstrategy). On the other hand, to enhance the purity of X sperm cellscollected in the stated population, it may be desirable to reject eachcoincident droplet containing a ˜X sperm cell even though the samedroplet may also contain an X sperm cell (coincident reject strategy).In general, as will be pointed out below, there are many controlstrategies which may be employed to maximize particle throughput andthere are many sorting strategies that may by employed with eachparticular control strategy. The strategies can be applied to varioussorting techniques using flow cytometry, such as droplet sorting,photo-damage sorting, and fluid-switching sorting. Further, the abovestrategies can be used to sort any type of particle according to anydesired characteristic or characteristics of the particle.

According to one embodiment, the microprocessor controls the rate atwhich the fluid delivery system delivers the fluid containing theparticles as a function of other variables of the system. For example,the microprocessor can control the fluid delivery rate as a function ofa desired output result. Since the microprocessor determines theidentity of each particle and determines whether such is directed to atleast one collected population, the microprocessor can determine andcontrol the output result by varying the control strategy and/or byvarying the sorting strategy. A desired output result may generally bedefined as at least one of the following: (1) the purity of at least onecollected population with respect to characteristic A particles orcharacteristic B particles (“high recovery”), and (2) the quantity ofcharacteristic A particles in the stated population relative to thetotal quantity of characteristic A particles in the stream, or thequantity of characteristic B particles in the stated population relativeto the total quantity of characteristic B particles in the stream (“highpurity”). As another example, the system may employ a substantiallyconstant fluid delivery rate and the microprocessor can control the sortparameters as a function of a desired output result. In this latterexample, the desired output result may generally be defined as acombination of (1) the purity of the particles in at least one collectedpopulation and (2) the quantity of desired particles available in thestream but not included in the stated population (“constant flow rate”).

In general, it may be assumed that when sorting two populations anidentified cell could have a 50/50 probability of being part of onepopulation or the other. However, it is also contemplated that anunidentified cell may in fact have some other probability other than a50/50 probability of being part of one population or the other. Thisother probability may be determined by empirical analysis or from othercharacteristics regarding the sample being sorted.

Several different control strategies are discussed in more detail below.

A. High Recovery Control Strategy

One type of control strategy may be referred to as a “high recovery”control strategy. The objective of this strategy is to maximize thenumber of desired particles sorted into the population of desiredparticles as long as the purity of that population is at or above anacceptable purity.

Pursuant to this strategy, the first particle sets described above aresorted into the population of desired particles because each of thesesets contains one or more particles having a desired characteristic A.The third particle sets are also sorted into the population of desiredparticles (coincident accept) because each of these sets also containsone or more particles having a desired characteristic A, albeitaccompanied by one more particles having characteristic B. On the otherhand, the second particle sets are rejected (i.e., not sorted into thepopulation of desired particles) because they do not contain a particlehaving the desired characteristic. To optimize throughput using thisstrategy, the microprocessor increases the fluid delivery rate as longas the purity of the collected population is at or above an acceptablelevel. Stated in the converse, the fluid delivery rate is increased aslong as the probable level of contamination of the population of desiredparticles is at or below an acceptable level.

As an example, consider the use of a high recovery control strategy forsorting X and Y sperm cells in the fluid stream of FIG. 78. The desiredresult may be to sort all of the X cells into a population of X cells solong as the population remains at or above an acceptable purity, e.g.,so long as X/(X+Y) is greater than 85% or some other acceptable level.To obtain this result, the first particles sets are sorted into apopulation of X cells because they contain only one or more X cells. Thethird particle sets are also sorted into the population of X cellsbecause they also contain one or more X cells, even though they may alsocontain a Y (or ˜X) cell. The second particle sets are sorted into someother population because they do not contain one or more X cells. Inthis example, the rate at which the fluid delivery system delivers thefluid containing the cells to the nozzle would continue to be increasedas long as the purity of the population of X cells is greater than 85%.Conversely, if the purity of the population of X cells falls below 85%,the fluid delivery rate is decreased.

In the context of a droplet sorting system, it is known from Poisson'sequation that for any given droplet generation rate, the number ofmultiple-cell droplets will increase as the cell delivery rateincreases. In other words, increasing the delivery rate of fluidcontaining the cells will increase the number of multiple-cell droplets.Therefore, if the coincident accept sorting strategy is used andcoincident droplets containing third particle sets are sorted into thepopulation of desired particles, increasing the fluid delivery rate willresult in a decrease in the purity of the collected population becauseat higher fluid delivery rates more coincident droplets are beinggenerated and collected. FIG. 79 illustrates this result for a two (2)particle fluid so that 100% of the desired particles are captured. Asshown, at very low fluid delivery rates (FDR along x axis), the purity(y axis) of the resulting collected population is very high because veryfew coincident droplets containing third particle sets are beinggenerated and collected. As the fluid delivery rate increases (FDRincreases to the right along the x axis), the percentage of coincidentdroplets generated increases resulting in more coincident droplets beingcollected and reducing purity of the usable population (along the yaxis). In the specific example illustrated, the fluid delivery rate is30K particles/second at about 80% purity.

The results of using a high recovery strategy can be dramatic, asillustrated by a simple example where X and Y sperm cells are sortedusing a droplet sorting process. Assume, for example that droplets aregenerated at a rate of 60K/sec, and that sperm cells are delivered tothe interrogation location at a rate of 30K/sec. According to Poisson'sequation, if all droplets containing X cells are sorted into thepopulation of X cells, including coincident droplets containing X and Ycells, about 15,000 X cells will be collected every second. Thecollected population will include about 2,600 Y cells, reducing thepurity of the population with respect to X cells to about 85.2%.However, the number of collected X cells (15,000) represents asubstantial increase relative to a strategy where coincident dropletsare not collected, as in the high purity strategy or mode discussedbelow. In the high purity mode, operating at a droplet frequency of40K/sec and cell delivery rate of 40K/sec (10K cells/sec more than inthe high recovery mode example above), only about 11,800 X cells arecollected every second, or about 3,800 X cells less than in the highrecovery strategy. Further, when the high purity strategy is used, about9,200 X cells are lost or wasted because coincident droplets are notsorted into the population of X cells. Therefore, if less than 100%purity is acceptable, it may be desirable to use the high recovery modeto increase the number of X cells collected or, stated conversely, todecrease the number of X cells lost.

In summary, in the high recovery control strategy using the coincidentaccept sorting strategy, the particle delivery rate is inversely relatedto the purity of the collected population of desired particles(sometimes referred to as the “usable” population).

B. High Purity Control Strategy

A second type of control strategy may be referred to as a “high purity”control strategy. The objective of this strategy is to maintain thepurity of the collected population with respect to particles having adesired characteristic at high level, so long as the quantity of desiredparticles in the collected population relative to the total number ofdesired particles available in the stream is at or above an acceptablequantity (i.e., so long as the quantity of desired particles in thestream which are not collected remains below an acceptable quantity).Pursuant to this strategy, the first particle sets described above aresorted into the population of desired particles because each of thesesets contains one or more particles having a desired characteristic A,and because these sets contain no contaminating particles. On the otherhand, the second and third particle sets are sorted into one or more“unusuable” populations (coincident reject) because they containcontaminating particles (i.e., characteristic B particles). To optimizethroughput using this “high purity” strategy, the microprocessorincreases the fluid delivery rate as long as the quantity of desiredparticles that are sorted into the usable population relative to thetotal number of desired particles available in the stream remains at orabove an acceptable quantity.

As an example, consider the use of a high purity control strategy forsorting X and Y sperm cells in the fluid stream of FIG. 78. The desiredresult may be to sort all of the X cells into a population of X cells solong as the quantity of X cells collected from the stream remains at orabove an acceptable quantity, e.g., at least 60%. To obtain this result,the first particles sets are sorted into the usable population becausethey contain only one or more X cells. On the other hand, the second andthird particle sets are sorted into one or more unusable populationsbecause they contain one or more contaminating (˜X) cells. In thisexample, the rate at which the fluid delivery system delivers the fluidcontaining the cells to the nozzle would continue to be increased aslong as the quantity of X cells collected in the usable population as apercentage of the total available quantity of X cells that have beensorted remains at or above 60%. Conversely, if the quantity of X cellsnot collected in the usable population rises above 40% of the totalnumber of available X cells that have been sorted, the fluid deliveryrate is decreased.

As noted above in the context of a droplet sorting system, it is knownthat increasing the fluid delivery rate will increase the number ofmultiple-cell droplets, and thus the number of coincident dropletscontaining third particle sets. Since coincident droplets are not sortedinto the population of collected X cells when using a coincident rejectsorting strategy, this means that increasing the fluid delivery ratewill result in an increase in the quantity of live X cells lost to theunusable population.

FIG. 80 illustrates this result for a two (2) particle fluid so that theusable population has 100% purity of desired particles. As shown, atvery low fluid delivery rates (FDR along x axis), the percentage ofdesired particles in the usable population is very high because very fewcoincident droplets are being generated and rejected. As the fluiddelivery rate increases (FDR increases to the right along the x axis),the percentage of coincident droplets containing third particle setsincreases and more such sets are rejected. This reduces the quantity ofdesired particles that are sorted into the usable population relative tothe total quantity of desired particles available in the stream (i.e.,the percentage of desired particles from the stream which are collectedin the usable population). In the specific example illustrated, thefluid delivery rate is about 40K particles/second and about 75% of thedesired particles are sorted into the usable population.

In summary, in the high purity control strategy implementing thecoincident reject sorting strategy, the particle delivery rate isinversely related to the percentage of desired particles in thecollected population (i.e., high purity of desired particles in theusable population).

C. Constant Flow Rate Control Strategy

A third type of control strategy may be referred to as a constant flowrate control strategy. In this strategy, the microprocessor maintainsthe fluid delivery rate constant (or within a constant range) and variesthe percentage of collected (or rejected) coincident droplets as long asthe purity of at least one collected population is at or above anacceptable level and as long as the quantity of desired particles inthat population is at or above an acceptable quantity relative to atotal quantity of desired particles that have been processed. Stated inthe converse, the fluid delivery rate is constant and the percentage ofaccepted (or rejected) coincident droplets varies as long as theprobable level of contamination of the usable population is at or belowan acceptable level of purity and as long as the probable quantity ofdesired particles that is lost to a population other than the stated(usable) population is at or below an acceptable quantity.

As an example, consider the use of a constant flow rate control strategyfor sorting the fluid stream shown in FIG. 78. The desired result may beto sort X sperm cells into a usable population having a purity of atleast 85% and to collect at least 60% of the X cells in the stream sothat no more than 40% of the X cells are sorted into the unusablepopulation. In this example, the rate at which the fluid delivery systemdelivers the particles would be maintained constant and the percent ofcollected or rejected third particle sets (coincident sets) would bevaried as long as the purity of the usable population with respect to Xcells is equal to or greater than 85%, and as long as the percentage ofX cells sorted into the unusable population is less than 40% so that thepercentage of desired particles sorted into the usable population isequal to or greater than 60% (variable coincident accept sortingstrategy). As the percentage of accepted third particle sets increases,the purity of the usable population decreases, but the quantity ofdesired particles (e.g., X cells) sorted into the unusable populationdecreases. Conversely, as the percentage of accepted third particle setsdecreases, the purity of the usable population increases, but thequantity of desired particles (e.g., X cells) that are sorted in theunusable population increases.

As noted above, it is known from Poisson's equation that the number ofmultiple-cell droplets (and thus the number of coincident dropletscontaining third particle sets) is constant for a constant fluid (cell)delivery rate. Since the number of coincident droplets is constant inthis control strategy, the percentage of coincident droplets sorted intothe usable population will impact both the purity of the usablepopulation and the quantity of X cells that are wasted by being sortedinto an unusable population. This is because the percent of unwanted Y(or ˜X) cells in coincident droplets which are accepted and sorted intothe collected unusable population is inversely related to the percent ofX cells in coincident droplets which are rejected and thus not sortedinto the collected usable population.

FIG. 81 illustrates the constant fluid delivery rate control strategy ina flow cytometry droplet sorting system and method as described hereinimplementing a variable coincident reject sorting strategy for a two (2)particle fluid. As shown, line OL illustrates the inverse relationshipbetween the percentage of rejected coincident droplets (x axis) comparedto the percentage of accepted coincident droplets (y axis). When thereis a very low percentage of accepted coincident droplets, there is avery high percentage of rejected coincident droplets. Conversely, whenthere is a very high percentage of accepted coincident droplets, thereis a very low percentage of rejected coincident droplets. Line OLillustrates this inverse relationship and represents the operating lineof the variable coincident accept sorting strategy at a given constantparticle flow rate. At point P in FIG. 81 along operating line OL, thepurity of the usable population is a given percentage depending onparticle flow rate, e.g., 85% purity. As the percentage of acceptedcoincident droplets increases (to the left and upward) along operatingline OL, the number of undesired particles that are sorted into theusable population increases and the purity drops below 85%, which may beunacceptable. As the percentage of accepted coincident dropletsdecreases (to the right and downward) along operating line OL, thepurity goes above 85% and is acceptable.

At point LL along operating line OL, 75% of the coincident droplets arerejected (i.e., sorted into the unusable population) so that thepercentage of desired particles that are wasted by being sorted into theunusable population is a given percentage based on the particle deliveryrate, e.g., 40%. As the percentage of rejected coincident dropletsincreases (to the right and downward) along operating line OL, thepercentage of desired particles that are sorted into the usablepopulation decreases (e.g., to <60%), which may be unacceptable. As thepercentage of rejected coincident droplets (to the left and upward)along operating line OL, the percentage of desired particles sorted intothe usable population increases (e.g., to >60%) and is acceptable. Thus,according to this aspect of the invention for a constant flow ratecontrol strategy implementing a variable coincident accept sortingstrategy, the microprocessor may operate the system so the percentage ofaccepted and rejected coincident droplets varies in an operating rangebetween point P1 and LL as indicated by arrow OR. Note that operatingrange OR may encompass more or less of the operating line, depending onthe level of tolerance for impurity and loss of desired particles to theunusable population.

In summary, in the constant flow rate control strategy using thevariable coincident accept sorting strategy, the percentage of thirdparticle sets which are accepted is inversely related to the purity ofthe usable population and inversely related to the quantity of desiredparticles wasted by being sorted to an unusable population.

D. Summary of Control Strategies

The following Table summarizes the control strategies noted above.

CONTROL HIGH HIGH CONSTANT STRATEGY RECOVERY PURITY FLOW RATE Controlledparameter Fluid delivery rate Fluid delivery rate Sort parametersControlling Purity of population Quantity of desired Purity ofpopulation AND parameter: particles in population Quantity of desiredparticles in population Sorting strategy Coincident accept Coincidentreject Variable coincident accept X/Y Sorting strategy Collect Xdroplets and Collect X droplets; Collect X droplets; vary X+~X droplets;reject ~X reject X+~X droplets percentage of collected X+~X droplets and~X droplets droplets; reject ~X droplets Definition The fluid deliveryrate is The fluid delivery rate The percentage of coincident increasedas long as the is increased as long droplets in the population is purityof the population as the quantity of increased as long as the puritywith respect to X particles desired particles in of the population withrespect is at or above an the usable population to X particles is at orabove an acceptable level relative to the total acceptable level; tocontinue quantity of X particles operation, the quantity of in thestream is at or desired particles in the usable above an acceptablepopulation relative to the total quantity quantity of X particles in thestream must be at or above an acceptable quantity Converse DefinitionThe fluid delivery rate is The fluid delivery rate The percentage ofcoincident increased as long as the is increased as long droplets in thepopulation is probability of as the probability of increased as long asthe contamination of the loss of the quantity of probability ofcontamination of usable population is at or desired particles in theusable population is at or below an acceptable level an unusable belowan acceptable level of of purity population is at or purity AND as longas the below an acceptable probability of loss of the quantity quantityof desired particles in the unusable population is at or Desiredresult >minimum acceptable >minimum >minimum acceptable purity purity;e.g., >85% purity acceptable quantity; and >minimum acceptablee.g., >60% of desired quantity; e.g., >85% purity and particlescaptured >60% of desired particles (<40% of desired captured (<40% ofdesired particles lost) particles lost)

Relatedly, a sorted sample obtained using one of the above controlstrategies can be combined with a second sample to obtain a final (e.g.,commercial) sample having the desired characteristics. For example, asample sorted according to the high purity strategy to produce a 100%pure population can be combined with a population of the same volumesorted to 80% purity to produce a final sample having a purity of 90%.Or in the case of animal sperm sorted to a high purity, an aliquotamount of the sorted sperm can be combined with an aliquot amount ofunsorted sperm to produce a final sample of desired purity at lower costthan sorting the entire amount of sperm using any of the above sortingmethods.

The above description of the control strategies assumes accurateidentification and sorting of each droplet including each coincidentdroplet. In practice, 100% accuracy is not possible for any number ofreasons. In order to minimize contamination, therefore, it may bedesirable to reject particles which cannot be classified with certaintyas belonging to the desired population. On the other hand, if certainparticles can be identified and classified as being in the desiredpopulation within a certain selected probability (e.g., greater than 50%in the case of sperm cells), it may be desirable to classify theparticles as belonging to the desired population so that they are notlost to the unusable population. Thus, as discussed earlier, particlessuch as sperm cells may be accepted or rejected for sorting into apopulation of desired cells based on the probability that such particlesbelong in the usable population.

The terms “usable” and “unusable” as used in the above table and thisapplication are used for convenience only and are not intended to belimiting in any way. Thus, a “usable” population includes any “first”population, regardless of how or whether it is used, and an “unusable”population includes any “second” population different from the usablepopulation, regardless of how or whether it is used. Similarly, a“desired” population means any population which is sorted according toselected particle characteristics.

The microprocessor and its signal processing software constitutes asystem for processing the electrical signals from the photodetector toclassify particles (e.g., particles in general and sperm particles inparticular) according to characteristics of the particles and to obtaininformation relating to the distribution of the particles as describedabove with respect to FIG. 78. Furthermore, the microprocessorconstitutes a control system responsive to the signal processingsoftware for varying the rate at which the fluid delivery systemdelivers particles to the nozzle system as a function of the obtainedinformation relating to the distribution of the particles. Furthermore,the microprocessor constitutes a control system responsive to the signalprocessing software for varying the sorting strategy as a function ofthe obtained information relating to the distribution of the particles.

In general, the microprocessor constitutes a control system responsiveto information received from the flow cytometry apparatus forcontrolling the sorting system to vary its sorting strategy or forcontrolling the fluid delivery system. In other words, themicroprocessor is capable of operating in a first mode to vary thesorting strategy, is capable of operating in a second mode forcontrolling the fluid delivery system, is capable of operating in athird mode to vary the sorting strategy and for controlling the fluiddelivery system, and may be capable of operating in other modes. Whenoperating in the first or third mode, the microprocessor is capable ofvarying the rate at which fluid is delivered as a function of at leastone of the following: (1) the purity of the at least one population withrespect to either characteristic A particles or characteristic Bparticles, and (2) the quantity of characteristic A particles orcharacteristic B particles in the at least one population relative tothe total quantity of characteristic A particles or characteristic Bparticles in the stream.

Collection System

A collection system is needed to collect the droplets after they passbetween the deflector plates. The collection system for a conventionalcytometer may be no more than collection vessels disposed to catch thedroplets in the various droplet streams after they pass between thedeflection plates. Similar conventional collection systems can be usedin some embodiments of the present invention.

However, it may be difficult to use a conventional collection system inembodiments of the present invention in which the nozzle is oriented todirect the fluid stream at an upward angle, thereby giving the dropletsa horizontal velocity component. One issue is that the droplets wouldtravel some horizontal distance along their arched trajectories beforethey begin downward movement that would be suitable for landing in acollection vessel. For example, if the nozzle is pointed upward at arange of 45° to 60° and the droplets exit at a velocity between 15 m/sand 20 m/s, the droplets will be a horizontal distance of several metersaway from the nozzle before they reach the apex of their trajectoriesand begin downward movement. Thus, a good deal of lab space would beoccupied by the droplet streams. Furthermore, at a range of severalmeters it could also be difficult to make sure the droplets land in theproper collection vessels. The trajectories of the droplet streams canchange whenever one or more operating conditions for the cytometerchange (e.g., adjustment to the fluid delivery rate resulting in achange in the fluid velocity at the nozzle orifice). Changes in thetrajectories of the droplet streams will be magnified by the distancethat the droplets travel. Thus, changes in the trajectories that do notresult in an appreciable change in droplet location at a pointrelatively near the nozzle could result in a significant change inlocation of the droplets at a location that is farther away from thenozzle. As discussed above, some embodiments of the present inventionemploy feedback to the droplet formation and/or sample fluid deliverysystems that could result in droplet streams that constantly alter theirtrajectories. One may also want to vary the pressure at which the sheathfluid is delivered to the nozzle. Air currents, temperature variations,and humidity variations could also alter the trajectories of the dropletstreams. Any factor that could change the trajectory of the dropletstreams could also require the collection vessels to be repositioned sothe droplets land in the appropriate collection vessels. In contrast,the trajectories of droplets streams in a conventional cytometer havinga downward pointing nozzle are less susceptible to variation. Forexample, the fact that the droplet streams have a substantially downwardinitial velocity means that variation in fluid velocity at the orificedoes not result in any significant variation in the trajectories.Furthermore, the collection vessels are relatively close to the nozzlewhich makes the collection system more tolerant to trajectory variationsin the droplet streams.

FIGS. 83-85 show one embodiment of a collection system, generallydesignated 2201, that may be used to collect sorted droplets in a systemof the present invention. The collection system is particularly suitedfor collection of droplets 33 when the cytometer nozzle system 101 isoriented to direct the fluid stream 21 at an upward angle or any otherangle having a horizontal component. As the droplets pass between thedeflector plates 629, they are sorted into multiple droplet streams 123,125 (e.g., two) having different arched trajectories. As shown in FIGS.84 and 85, the trajectory of each droplet stream leads to one of twodroplet intercepting devices 2203. If the droplets are being sorted intomore than two droplets streams, a separate intercepting device wouldneed to be provided for each additional droplet stream. Thus, the numberof intercepting devices in a collection system of the present inventionwill depend on the number of streams into which the droplets are beingsorted.

Each intercepting device 2203 in the exemplary collection system has animpact surface 2205 positioned to span the trajectory of one of thedroplet streams to divert droplets moving along that trajectory to acollection vessel 2207 positioned beneath each intercepting device. Theimpact surfaces are preferably made of a pliable material. Without beingbound by a particular theory, it is believed that pliable materialscushion the impact of droplets striking the surface of the interceptingdevice, thereby reducing damage to the particles (e.g., sperm cells) inthe droplets. For example, the intercepting devices may be constructedof polypropylene, polyethylene, or other similar polymers. Referring toFIGS. 86 and 87, the intercepting devices 2203 have been constructed bycutting a droplet entryway 2211 (e.g., rectangular window) in one sideof the bulb 2213 of a pipette 2215. Thus, a portion of the inside wall2217 of the bulb opposite the droplet entryway forms a curved impactsurface 2205 which spans the trajectory of the respective dropletstream. Conveniently, the tube of the pipette serves as a guide 2225 fordirecting fluid from the impact surface to the collection vessel.

Referring to FIG. 84, the intercepting devices are fastened to acollection system frame 2227, which holds them in place. In order toaccount for variability in the trajectories of the droplet streams, itis desirable to allow adjustment of the positions of the interceptingdevices. For example, the vertical height of each intercepting devicemay be adjusted by sliding the guide tube up and down in a circular borethrough a holder 2229. When the intercepting device is at the desiredheight, a set screw 2231 may be tightened to hold it at that height. Theholder may be attached to a mounting plate 2233 which is attached to thecollection system frame, for example by set screws 2235 (e.g., two setscrews). The set screws 2235 pass through a horizontal slot 2241 in themounting plate to allow lateral adjustment of the intercepting device.After adjustment, the set screws may be tightened to hold the circularholder in the desired location. Those skilled in the art will recognizethat a variety of other fastening devices could be used to adjust theposition of the intercepting device without departing from the scope ofthe present invention. The collection vessels 2207 are held beneath theintercepting devices in slots 2241 in a tray 2243 for holding thecollection vessels. Thus, each collection vessel may be moved within arespective slot as necessary for it to remain in position under therespective intercepting device. Also, a water bath (not shown) may beprovided around the collection vessel if desired to control thetemperature of the contents of the collection vessel.

Referring to FIG. 85, in one embodiment of the present invention an exitwindow 2245 (e.g., a rectangular window) has been cut in the back of oneof the intercepting devices 2247 to allow one or more droplet streams topass through the intercepting device. A second intercepting device 2249is positioned behind the exit window to intercept the droplets that passthrough this window. An exemplary entry window for the secondintercepting device may be approximately the same size as the exemplaryexit window for the first intercepting device. For reasons that will beapparent, it is desirable for the exit window to be significantlysmaller than the entry window for the first intercepting device. Forinstance, an exemplary entry window for the first intercepting device isabout ⅝ of an inch high and about ⅜ of an inch wide. In contrast, anexemplary exit window may be ⅛ of an inch high and 5/16 of an inch wide.

During operation of the cytometer, the collection system operates tointercept the droplets in the sorted streams. The intercepted dropletsthen drain down through the guide tubes 2225 of the intercepting devices2203 and into the collection vessels 2207. In a case in which acytometer has an upward pointing cytometer nozzle that directs dropletstreams along arched trajectories, for example, the intercepting devicesallow the droplets to be intercepted at a point on their trajectory thatis significantly closer to the nozzle in comparison to the point atwhich the droplets would be collected by a conventional collectionsystem (i.e., a collection system without intercepting devices).

Intercepting the droplet streams relatively early along their archedtrajectories (e.g., while they are still moving upward) reduces theamount of variation in the location of the droplets at the time thedroplets first encounter the collection system. Accordingly, thecollection system can tolerate more variation in the trajectories of thedroplet streams than a convention collection system could tolerate.Similarly, the droplets are less likely to be buffeted by air currentsbecause of their shorter paths to the collection system.

A balance must be struck between moving the intercepting devices 2203closer to the nozzle 101 to increase tolerance for trajectory variationsand moving the intercepting devices farther away from the nozzle orificeto reduce or minimize the force of impact when droplets impact theintercepting devices, as by positioning the intercepting devices so theyintercept the droplet streams substantially at the apex of theirtrajectories. Accordingly, the best location for the interceptingdevices will depend on the durability of the particles (e.g. spermcells) being sorted, the droplet velocities, and the expected magnitudeof variation in the droplet stream trajectories. In the case of dropletscontaining bovine sperm cells having a velocity at the nozzle orifice ofabout 16 to 20 m/s, for example, the intercepting devices may bepositioned in the range of 4-6 inches from the nozzle orifice. In theembodiment in which a first intercepting device has an exit window and asecond intercepting device is positioned behind the first interceptingdevice, for example, the first intercepting device may be in the rangeof about 4 and 5 inches from the nozzle. More desirably, the firstintercepting device is about 4.5 inches from the nozzle. The secondintercepting device may be in the range of about 5 to 6 inches from thenozzle. More desirably, the second intercepting device is about 5.5inches from the nozzle.

The configuration in which one intercepting device 2203 is positioned tointercept the droplets that pass through an exit window of anotherintercepting device is particularly advantageous when one is notconcerned about the purity of one of the sorted populations (e.g., Ychromosome-bearing sperm in the case of sperm sorted for use in breedingdairy cattle). Those skilled in the art will know that a number of straydroplets 2265 (e.g., a mist of stray droplets) having unknown contentsmay be produced by the cytometer in addition to the droplets in thesorted streams as shown in FIG. 85. The first intercepting device shouldbe positioned so that the stream of droplets that are being sorted intothe population for which there is the greatest tolerance for impuritieshit the impact surface 2205 of the first intercepting device and thestream for which purity is most critical passes through the exit window2245 to hit the impact surface of the second intercepting device. Thisway the majority of the stray droplets are collected into the collectionvessel 2207 containing the population for which there is less concernabout purity, as shown in FIG. 85, and will not contaminate thepopulation for which purity is critical. Also by intercepting andcollecting the stray droplets, one avoids the need to clean as often asif the stray droplets escape the collection system. In contrast to thefirst intercepting device, the second intercepting device only divertsdroplets that pass through the smaller exit window. This facilitatesmaintenance of the purity of the population collected by the secondintercepting device.

Those skilled in the art will recognize that the exemplary collectionsystem could readily be modified in a number of ways without departingfrom the scope of the present invention. For example, it would bepossible to construct a droplet intercepting device having an integrallyformed (or otherwise attached) collection vessel beneath it, withoutdeparting from the scope of this invention. Similarly, although theintercepting devices in the embodiment shown in FIGS. 83-87 are modifiedpipettes, it is understood that the intercepting devices 2203 can be anyof a variety of shapes without departing from the scope of thisinvention. For example, each intercepting device may comprise a flat orcurved sheet, a spoon, a bowl, or other similar shape. The onlyrequirement is that the intercepting device is operable to interceptdroplets moving along a respective trajectory of a droplet stream and todivert the intercepted droplets into a collection vessel. However, oneadvantage to constructing the intercepting devices out of a readilyavailable and relatively inexpensive product, such as a pipette, is thatit may be more economical to replace and dispose of the usedintercepting devices after each sample run rather than clean theintercepting devices between sample runs. This could help reduce costsof operating the collection system.

Collection Fluid

The sorted sperm are collected in a vessel that contains a collectionfluid 2301 (FIGS. 56 and 57). Generally, the purpose of the collectionfluid includes cushioning the impact of the sperm cells with thecollection vessel or providing a fluid support for the cells. Consistentwith these considerations, the collection fluid may comprise a buffer orbuffered solution and a protein source.

If included, examples of buffers or buffered solutions that may be usedin the collection fluid are disclosed above with respect to samplecollection and dilution. Typically, these buffers or buffer solutionswill be in a concentration of about 0.001M to about 1.0M and have a pHof about 4.5 to about 8.5, preferably of about 7.0. In one embodiment,the collection fluid contains buffer comprising 0.96% Dulbecco's PBS(w/v) at a pH of about 7.0 In another embodiment, the collection fluidcontains a metabolic inhibitor comprising 0.204 g NaHCO₃, 0.433 g KHCO₃,and 0.473 g C₆H₈0₇H₂O per 25 mL of purified water (0.097 moles/L ofNaHCO₃, 0.173 moles/L of KHCO₃, 0.090 moles/L C₆H₈0₇H₂O in water).

If included, the protein source may be any protein source that does notinterfere with the viability of the sperm cells and is compatible withthe particular buffer or buffered solution being used. Examples ofcommon protein sources include milk (including heat homogenized andskim), milk extract, egg yolk, egg yolk extract, soy protein and soyprotein extract. Such proteins may be used in a concentration from about1% (v/v) to about 30% (v/v), preferably from about 10% (v/v) to about20% (v/v), and more preferably about 10% (v/v). While milk may be usedin combination with a buffer or buffered solution, generally milk isused in the absence of the same, as milk is a solution itself that mayserve the same purpose of a buffer or buffered solution. In suchinstances, the collection fluid may contain about 80% (v/v) to about 90%(v/v) milk.

In addition to or in lieu of the protein source, the collection fluidmay also comprise seminal plasma. Seminal plasma serves the dualbenefits of improving sperm viability and motility and of stabilizingthe sperm membrane (thereby preventing capacitation during thecollection and storage of the sperm). Maxwell et al., Reprod. Fert. Dev.(1998) 10: 433-440. The seminal plasma may be from the same mammal fromwhich the semen sample was obtained, from a different mammal of the samespecies, or from a mammal of a different species. If included in thecollection fluid, typically the percentage of seminal plasma will be inthe range of about 0.5% (v/v) to about 10% (v/v). If used in combinationwith a protein source, such as for example egg yolk or milk, the totalpercentage of seminal plasma and protein source will range from about 1%(v/v) to about 30% (v/v). In such instances, the percentage of seminalplasma will be inversely proportional to the percentage of the proteinsource. Accordingly, in one embodiment, the collection fluid comprisesseminal plasma. In another embodiment, the collection fluid containsseminal plasma in an amount of about 0.5% (v/v) to about 10% (v/v),preferably in an amount of about 4% (v/v) to about 6% (v/v), and morepreferably in an amount of about 5% (v/v). In another embodiment, thecollection fluid contains a protein source and seminal plasma. In yetanother embodiment, the collection fluid comprises seminal plasma andegg yolk, the percentage of both totaling between about 1% (v/v) andabout 30% (v/v).

Optionally, the collection fluid may also contain a range of additivesthat are beneficial to sperm viability or motility. Examples of suchadditives include an energy source, an antibiotic, and a compositionwhich regulates oxidation/reduction reactions intracellularly and/orextracellularly, each of which is discussed above with respect to samplecollection and dilution. Such additives may be added to the collectionfluid in accordance therewith.

Accordingly, in a certain embodiment, the collection fluid comprises0.96% Dulbecco's PBS (w/v), 1% (w/v) fructose, 10% (v/v) egg yolk inwater, at a pH of about 7.0. In yet another embodiment, the collectionfluid further comprises 10 mM pyruvate, 100 □M vitamin K, or 1 mM oflipoic acid.

Alternatively, and in lieu of the use of a collection fluid, the sortedcells may be collected into a vessel containing or coated with acryoextender used in the subsequent cryopreservation steps and furtherdescribed below. Accordingly, in one particular embodiment, the sortedcells are collected into a cryoextender. In another embodiment, thecollected cells are sorted into a cryoextender comprising water,Triladyl® (Minitube, Verona, Wis., comprising glycerol, tris, citricacid, fructose, 5 mg/100 ml tylosin, 25 mg/100 ml gentamycin, 30 mg/100ml Spectinomycin, and 15 mg/100 ml Lincomycin), egg yolk, and pyruvicacid. In yet another embodiment, the collection fluid is thecryoextender comprising 25 g Triladyl®, 25 g egg yolk, and 10 mM pyruvicacid in 75 mL of water.

It is to be understood that the percent concentrations of protein in thecollection fluid disclosed herein are those prior to the addition of theflow sorted cells. The addition of the flow sorted cells will dilute thefinal concentration of the collection fluid to about 1/20 that of whatit was prior to the addition of the flow sorted cells. Therefore, forexample, the collection fluid may initially contain about 10% (v/v) eggyolk. After the flow sorted cells are collected in the collection vesselcontaining the collection fluid, the final concentration of egg yolkwill be reduced to about 0.5% (v/v).

Pre-Treatment of Intercepting Devices and/or Collection Vessels

In order to minimize possible damage to particles (e.g., sperm cells)that may be sorted according to the present invention, the interceptingdevices 2203 and/or collection vessels 2207 (FIGS. 56-60) may be treatedprior to use. Such pre-treatment may comprise, for example, contactingor soaking the intercepting devices and collection vessels in a bathcontaining a composition that will serve to minimize the impact betweenthe particle and the intercepting device. Upon removal of theintercepting devices and collection vessels from the bath, a certainamount of the composition will remain on the intercepting devices andcollection vessels and serve as a cushioning agent for the particles inthe droplets. The composition, therefore, should have characteristicssuitable for providing the desired cushioning effect. In addition, thecomposition should also be compatible with the particle or cell beingsorted, the sheath fluid, and the collection fluid. Consistent withthese considerations, the composition used to treat the interceptingdevices and collection vessels may comprise a buffer or bufferedsolution, a sheath fluid, a collection fluid, a cryoextender, anycomponents contained in the buffered solution, sheath fluid, collectionfluid, or cryoextender, or any combination thereof. Buffers, bufferedsolutions, sheath fluids, and collection fluids used for the stainingand separation of sperm cells according to the methods of the presentinvention are described above. Accordingly, in one embodiment, theintercepting devices and collection vessels are contacted with (e.g.,soaked in or brushed with) sheath fluid. In another embodiment, theintercepting devices and collection vessels are contacted withcollection fluid. In yet another embodiment, the intercepting devicesand collection vessels are contacted with a cryoextender describedbelow.

The contacting or soaking of the intercepting devices and collectionvessels with the composition preferably occurs for a period of timesufficient to allow the composition to adhere to the surfaces of theintercepting devices and collection vessels. Such a period of time isgenerally less than about 90 minutes, preferably less than about 60minutes, more preferably about 30 to about 60 minutes, and mostpreferably about 60 minutes. In still another embodiment, theintercepting devices and collection vessels are merely contacted withthe composition prior to use.

In lieu of or in combination with the contacting of the interceptingdevices and collection vessels with the above-described composition, theintercepting devices and collection vessels may also be contacted withspecific components contained in the sheath fluid, the collection fluid,and/or the cryoextender, such as for example, BSA, SSS, egg yolk, eggyolk extract, milk (including heat homogenized and skim), milk extract,soy protein, and soy protein extract. Accordingly, in one embodiment,the intercepting devices and collection vessels are contacted withsheath fluid and subsequently contacted with 0.1% (v/v) bovine serumalbumin. In another embodiment, the intercepting devices and collectionvessels are contacted with sheath fluid and subsequently contacted with10% (v/v) egg yolk. In another embodiment, the intercepting devices andcollection vessels are soaked in collection fluid and subsequentlycontacted with 0.1% (v/v) bovine serum albumin. In another embodiment,the intercepting devices and collection vessels are soaked in collectionfluid and subsequently contacted with 10% (v/v) egg yolk.

Although the intercepting devices and collection vessels receive thesame pre-treatment in each embodiment described above, it is possible touse different pre-treatment protocols for the intercepting devices andthe collection vessels without departing from the scope of thisinvention. Likewise, some of the intercepting devices or collectionvessels could receive one pre-treatment and others of the interceptingdevices or collection vessels could receive a different pre-treatmentwithout departing from the scope of this invention. Certain advantagesof the pre-treatment can also be obtained by pre-treating only theintercepting devices or only the collection vessels, again withoutdeparting from the scope of this invention.

Concentration

As noted above, the sorted sperm collected by the flow cytometer havebeen diluted by the addition of various buffers and extenders, thestaining fluid, the sheath fluid, and the collection fluid. Typically,the concentration of sperm cells after sorting by flow cytometry asdescribed above is in the range of about 0.7-1.4×10⁶ sperm cells/ml.Therefore, it is important to concentrate the sorted sperm cells tominimize the dilution shock to the sperm and to attain the properconcentration of sperm for cryopreservation and artificial insemination.Standard practice in the animal breeding industry, for example, is toperform artificial insemination with sperm at a concentration of eitherabout 20×10⁶ or about 40×10⁶ sperm cells/ml. One way to concentrate thesperm cells is through centrifugation of the fluid collected by thecytometer. Another way to concentrate the sperm is to pass the fluidcollected by the cytometer through a filtration system. These methodsare discussed in more detail below.

A. Centrifugation

Any conventional centrifuge can be used to concentrate sperm. However ina commercial operation it is preferable to use a centrifuge having thecapacity to centrifuge a large batch of sperm cells at once. Duringcentrifugation a majority of the sperm cells will collect in a pellet atthe bottom of the centrifuge tube due to the centrifugal force acting onthe sperm cells. The magnitude of the centrifugal force isconventionally stated as the number of times the centrifugal forceexceeds the gravitational force. Because the centrifugal force is thecritical parameter and because the magnitude of the centrifugal force atany given speed (angular velocity) will vary depending on the length ofthe radius of curvature, the speed of centrifugation is typicallyspecified by stating the magnitude of the centrifugal force. Forexample, a 600 g force means the angular velocity of the centrifuge isselected so the resulting centrifugal force will be 600 times the forceof gravity. The majority of the fluids and any sperm cells that escapebeing centrifuged into the pellet will be in the supernatant. Generally,the supernatant is removed and the sperm cells in the pellet areresuspended for further processing as described below. It is importantto maximize the percentage of sperm that are concentrated in the pellet,while at the same time minimizing damage to the sperm cells.

According to one method of the present invention, a centrifuge tubecontaining about 10×10⁶ sorted sperm cells is placed in a centrifuge. Tofacilitate concentration, centrifuge tubes may be used as the collectionvessels in the collection system of the cytometer. This avoids the needto transfer the sorted sperm cells to a centrifuge tube beforecentrifugation. The tube is centrifuged at a speed and for a durationthat is sufficient to cause a pellet of concentrated sperm cells to formin the bottom of the tube. The speed and duration of the centrifugationis desirably selected in consideration of several factors, including:the fact that sperm cells are fragile and can be damaged bycentrifugation at an excessive speed; the size of the centrifuge tubewill affect the time required for sperm cells to move to the bottom ofthe tube; and the sperm cells are more likely to be damaged bycentrifugation at a given speed the longer the centrifugation continues.Thus, in one embodiment of the present invention the centrifuge tube iscentrifuged at 550-800 g for a period of about 6-10 minutes. Accordingto another embodiment of the present invention, the centrifuge tube iscentrifuged at 650-750 g for a period of about 6-10 minutes. In stillanother embodiment, the centrifuge tube is centrifuged at 700 g for aperiod of about 6-10 minutes. In yet another embodiment, the centrifugetube is centrifuged at 700 g for a period of about 7 minutes.

As demonstrated in the following experiments, the speed of thecentrifuge and the duration of centrifugation may affect the percentageof sperm cells recovered and the motility of the recovered sperm cells.The experiments were conducted without actually sorting the sperm cells.Instead, various fluids including buffers, extenders, sheath fluids anda staining fluid were added to semen samples to simulate the sortingprocess. The samples were then centrifuged in an attempt to concentratethe sperm cells.

Centrifuge Example 1

In centrifuge example I bovine semen was collected and evaluated asdescribed above. The semen sample was diluted with a quantity ofTris-citric acid (“TCA”) having a pH of 7.3 to attain a concentration of150×10⁶ sperm cells/ml. Spermatozoa were stained with Hoechst 33342(100, μM) at 41° C. for twenty minutes. Two 15 ml tubes were preparedwith buffers for the simulation. Tube 1 was partially filled with 750 μlof phosphate buffered saline (“PBS”) with 10% egg yolk and 14.25 ml PBSwith 0.1% bovine serum albumin (“BSA”). Tube 2 was partially filled with750 μl TCA with 10% egg yolk and 14.25 ml PBS with 0.1% BSA. Each of thetwo tubes received 100 ul of the solution containing the stainedspermatozoa, which were then incubated at room temperature for 20minutes. The two tubes were then divided into two aliquots of 7 ml each.One aliquot from each tube was centrifuged at 2250 rpm (about 540 g) for7 minutes in a fixed bucket centrifuge. The other aliquot from each ofthe two tubes was centrifuged at 2500 rpm (about 660 g) for 7 minutes.Immediately after centrifugation, 10 ml pipettes were used to remove andsave the supernatant from each aliquot. The pellets were resuspended in200 ul of TCA with 10% egg yolk (pH 7.0). Pre- and post-centrifuge spermmotility was observed under a phase contrast microscope. Fifty ul of afixative (0.1% glutarldehyde in 3.4% Na citrate) was added to eachpellet and supernatant to immobilize the sperm for concentrationdetermination with a hemacytometer. Total numbers of spermatozoa werecalculated on the basis of volume used/recovered multiplied by thecorresponding sperm concentration as determined by the hemacytometer.The recovery rate was calculated as the total number of sperm in thepellet divided by the sum of the total number of sperm in the pellet andthe total number of sperm in the supernatant.

The results, as shown in FIGS. 88 and 89 show there is little differencein sperm cell motility caused by varying the centrifuge speed. Theresults also show that motility was slightly better using TCA comparedto PBS.

Centrifuge Example II

In centrifuge example II semen samples from three bulls were collectedand evaluated as described above. One of the samples was disqualifiedfor failure to meet initial quality control standards. The other twosemen samples were diluted with a quantity of TCA having a pH of 7.3 inorder to obtain a sperm concentration of 150×10⁶ sperm/ml. Thespermatozoa were stained with a 10 μM Hoechst 33342 solution at 41° C.for twenty minutes. A simulated buffer containing 1500 μl PBS with 10%egg yolk and 28.3 ml PBS with 0.1% BSA was added to each of two tubes.Two hundred μl of the stained spermatozoa (30×10⁶ sperm cells) wereadded to each tube and incubated at room temperature for twenty minutes.Three 9 ml aliquots of semen mixture were taken from each of the twotubes for centrifugation. One aliquot from each of the two samples wascentrifuged for seven minutes in a 15 ml centrifuge tube at each of thefollowing speeds: 550 g; 650 g; and 750 g. The temperature duringcentrifugation was 22° C. Immediately after centrifugation, supernatantwas removed with a 10 ml pipette, leaving about 200-300 μl supernatantin the pellet. The pellets were resuspended with 200 μl of TCA having10% (v/v) egg yolk having a pH of 7.0. Pre- and post-sort sperm motilitywas observed under a phase contrast microscope. Severe spermagglutination was noted in the post-centrifuge samples from one of thetwo bulls. Fifty μl of a fixative (0.1% glutardehyde in 3.4% Na citrate)was added to each supernatant and pellet to immobilize the sperm forconcentration determination. Recovery rate was determined according tothe formula set forth in centrifuge experiment 1.

The results are shown in FIG. 90. The results show improved recoveryrate of sperm cells at 650 g compared to 550 g. However, there waslittle difference in recovery rate between 650 g and 750 g. There was nosignificant difference in sperm cell motility caused by varying thespeed of the centrifuge.

Centrifuge Example III

For centrifuge example III, the procedure of centrifuge example II wassubstantially repeated with the same three bulls on a different day. Theresults are shown in FIG. 91. The results confirm that there is littledifference in the recovery rate at 650 g compared to 750 g.

Centrifuge Example IV

Semen was collected from two different bulls on two different days.Semen was transported and evaluated in the manner described above. Basedon sperm concentration of raw semen, spermatozoa were diluted withTris-citric acid (TCA, pH 7.3) plus 10 mM pyruvate, to a concentrationof 150×10⁶ sperm/ml. The spermatozoa were stained with 10 μM Hoechst33342 at 41° C. for 20 min. After staining, 267 μl of the solutioncontaining the stained spermatozoa were diluted to a concentration of1×10⁶ sperm/ml by addition of the following simulated buffers: 2 ml PBSwith 10% (v/v) egg yolk; and 37.733 ml PBS with 0.1% (w/v) bovine serumalbumin (BSA). The stained spermatozoa and simulated buffers wereincubated at room temperature for at least 20 minutes. Four 9 mlaliquots were taken from the stained spermatozoa and simulated buffermixture obtained from each bull. The four aliquots from the first bullwere centrifuged at varying combinations of centrifuge speed andduration in the following sequence:

(1) 700 g for 7 minutes for the first aliquot;

(2) 700 g for 10 minutes for the second aliquot;

(3) 650 g for 10 minutes for the third aliquot; and

(4) 650 g for 7 minutes for the fourth aliquot.

The four aliquots from the second bull were centrifuged at varyingcombinations of centrifuge speed and duration in the following sequence:

(1) 700 g for 10 minutes for the first aliquot;

(2) 700 g for 7 minutes for the second aliquot;

(3) 650 g for 10 minutes for the third aliquot; and

(4) 650 g for 7 minutes for the fourth aliquot.

All centrifugation was performed in 15 ml centrifuge tubes in a swinghead centrifuge (Allegra 6R, Beckman Coulter Inc. Fullerton, Calif.) at22° C. The time interval between semen collection at farm andcentrifugation in lab was 4-5 hours. Immediately after centrifugation,supernatant was removed with 10 ml pipettes, leaving ˜250 μl supernatantwith each pellet. The pellets were resuspended in 250 μl of Delbecco'sPBS (pH 7.0). Sperm motility and progressive motility were observedusing a Hamilton-Thorn Motility Analyzer (two slides per sample; twochambers per slide) after staining but before centrifugation and againafter centrifugation. Sperm concentration was determined byhemacytometer measurement of a 100 μl aliquot of the pre-centrifugestained spermatozoa and simulated buffer mixture that had been placed inthe freezer and a 10 μl aliquot of the resuspended pellet mixed with 90μl fixative (0.1% glutaraldehyde in 3.4% Na citrate). Recovery rate wasdetermined as in Centrifuge Sample 1. The results are shown in FIGS. 92and 93.

The data indicate that >85% of the spermatozoa can be recovered aftercentrifugation at 650 g or 700 g, for 7 or 10 minutes (FIG. 92).However, recovery rate was slightly better (95%) at 700 g. The declinein motility after centrifugation (compared to before centrifugation) inall treatments could be due to the presence of dead/abnormal/fragilespermatozoa which could not withstand the stress of centrifugal force.Sperm motility declined by 10-14% (FIG. 93) in all treatments. Thehigher decline in sperm motility (14%) at 650 g for 7 min might be dueto the longer exposure of sperm to simulated buffer as centrifugation at650 g was conducted after 700 g. Centrifugation did not show any adverseeffect on progressive motility of spermatozoa, rather there wasimprovement by 2-3%.

Centrifuge Example V

Semen was collected from one bull on two different days. Semen wasevaluated, diluted and stained with Hoechst 33342, and further dilutedin simulated buffers as described in Centrifuge Example IV. Four 9 mlaliquots of the stained spermatozoa and simulated buffer mixture wereobtained for each of the two semen samples. The aliquots from the firstsample were centrifuged at one of the following combinations ofcentrifuge speed and duration in the following sequence:

(1) 750 g for 10 minutes for the first aliquot;

(2) 750 g for 7 minutes for the second aliquot;

(3) 700 g for 10 minutes for the third aliquot; and

(4) 700 g for 7 minutes for the fourth aliquot.

For the aliquots obtained from the second sample, the combinations ofcentrifuge speed and duration were the same, but the sequence wasmodified as follows:

(1) 750 g for 7 minutes for the first aliquot;

(2) 750 g for 10 minutes for the second aliquot;

(3) 700 g for 7 minutes for the third aliquot; and

(4) 700 g for 10 minutes for the fourth aliquot.

Centrifugation was conducted in a 15 ml centrifuge tube in a swing headcentrifuge (Allegra 6R, Beckman Coulter Inc. Fullerton, Calif.) at 22°C. The interval between semen collection at farm and centrifugation inlaboratory was about 6.5 hours for the first sample, and about 4 hoursfor the second sample. Post centrifugation processing, i.e. removal ofsupernatant, resuspension of pellet, determination of spermconcentration, and motility estimation via Hamilton-Thorn MotilityAnalyzer, were conducted following the same procedure as described inExample IV. The results are shown in FIGS. 94 and 95.

The results show that >85% of the sperm population in highly dilutedsuspension can be recovered with 700 g or 750 g in 7 minutes or 10minutes (FIG. 94). An increase in g force to 750 g did not improve therecovery rate significantly. As was the case in Centrifuge Example IV,the decline in motility after centrifugation (as compared to beforecentrifugation) was observed in all treatments. In the presentexperiment, sperm motility declined by 13-20% (FIG. 95) which is littlehigher than in Centrifuge Example IV. The variation could be due tovariation in semen sample and longer time interval from semen collectionto centrifugation (6 hours) in one replicate. As explained in ExampleIV, the decline in sperm motility (about 20%) at low speedcentrifugation (700×g, for 7 or 10 min) might be due to the longerexposure of sperm to simulated buffer as they were centrifuged after 750g centrifugation. The decline in progressive motility was negligible(1-5%).

B. Secondary Centrifugation

In order to recover sperm that might otherwise be lost in thesupernatant, it is possible to centrifuge the supernatant after it hasbeen separated from the pellet. Without being bound by a particulartheory, applicants believe the pellet/supernatant interphase impedesmovement of spermatozoa into the pellet. Removal of the interphase byseparating the pellet from the supernatant will allow furthercentrifugation of the supernatant to cause sperm cells that would haveremained in the supernatant to form a second pellet. The second pelletcan be resuspended and added to resuspended sperm from the first pellet.

C. Filtration

An alternative concentration method that may be used to avoid loss ofsperm cells in the supernatant is filtration. As shown in FIG. 96,according to one exemplary embodiment a filter 2415 is incorporated in acollection vessel 2403. The size of the pores in the filter aredesirably in the range of about 0.2-1 microns. It is also desirable thatthe filter is not a depth filter (e.g., a filter having tortuouspassages in which sperm tails can be caught). Rather it is desirablethat the filter be as thin as possible. For example, it is desirablethat the filter thickness be in the range of 50 μm to 500 μm; moredesirable that the filter thickness be in the range of 75 μm to 250 μm;and most desirable that the filter thickness be in the range of 100 μmto 150 μm. A low level vacuum 2417 is applied to remove the fluidsthrough the filter as the droplets 33 are collected. It is important touse a low level vacuum (less than 20 inches of mercury, e.g., 15 inchesof mercury) to avoid inflicting damage to the sperm cells. In oneembodiment the vacuum is low enough that the fluid removal rate is about1.0 ml/15 seconds. According to another embodiment of the presentinvention, the vacuum is applied intermittently to allow the sperm cellsa chance to recover. In still another embodiment, the filter 2415 isconstructed of a material that is compatible with sperm cells, yet hasno binding affinity for them. At the completion of the sort, about80-90% of the fluids will have been removed through the filter. However,enough fluid remains (about 10-20%) that the sperm cells are in aconcentrated slurry 2405, thereby preventing the sperm cells fromforming a filter cake. The concentrated suspension may be transferred toanother container 2419, as shown in FIG. 97 for example. A syringemechanism 2409 with a cannula-tip filter 2411 can be used to remove someof the remaining liquid from this container 2419. However, enough fluidsare left in the container to prevent the sperm cells from caking on thefilter 2411. The same considerations apply to the cannula tip filter2411 as the filter 2415 in the collection vessel. Thus, the cannulafilter 2411 pore size is desirably in the range of about 0.2-1.0 micronsand the cannula filter is relatively thin to avoid having sperm tailsgetting caught in tortuous passages in the filter. For example, aDynaGard® hollow polypropylene fiber syringe tip filter, which iscommercially available from Spectrum Laboratories, Inc. of RanchoDominguez, Calif. may be used for the cannula tip filter. As shown inFIG. 98, a resuspension fluid 2413 is flushed through the cannula-tipfilter to wash cells that may be sticking to the filter surface backinto the slurry. The resuspension fluid may include a quantity of thefiltered fluid and/or a suitable extender. After a quantity ofresuspension fluid sufficient to remove sperm cells from the filter hasbeen back flushed through the filter, additional resuspension fluid maybe added if desired. The total quantity of resuspension fluid isselected to bring the concentration to a desired concentration (e.g.,about 20×10⁶ sperm cells/ml). Thus, the filtration process of thisembodiment is a three-step process involving the use of a filter in thecollection vessel, filtration using a cannula filter, and resuspensionto obtain the desired concentration.

In an alternative two-step filtration process, the first and secondsteps of the three-step process described above are combined so thatremoval of all fluid is through a cannula filter. In this process thesorted sperm cells are directed to a collection vessel that does nothave a filter. The fluids are removed by low vacuum and/or intermittentvacuum as described above that is applied through the cannula-tip filter2411. When the sperm cells are in a concentrated slurry, a resuspensionfluid, such as for example, an extender, is flushed back through thecannula filter to obtain the desired concentration of sperm cells.

Filtration Example I

Filtration example I shows the recovery rate and motility of sperm cellsafter concentration by a three-step filtration process of the presentinvention. Semen samples were collected from three bulls and evaluatedas provided in the sample preparation section above. One of the threesemen samples was disqualified for failing to meet minimum initialquality criteria. Two remaining samples were diluted with a quantity ofTCA (pH 7.3) necessary to attain a concentration of 150×10⁶ spermcells/ml. Five hundred ul PBS with 10% egg yolk and 9.5 ml PBS with 0.1%BSA was added to each of two 15 ml test tubes. Sixty-seven ul of semensample (about 10×10⁶ sperm cells) was added to each test tube andincubated for twenty minutes at room temperature. Referring to FIG. 99,a vacuum pump 2427 was used to apply negative pressure to draw a four mlaliquot of the diluted semen 2423 through a filter 2425. The filtrate2429 was collected in a syringe 2421. After filtration sperm cells onthe filter were flushed back with 1 ml TCA buffer in a 15 ml tube. Spermmotility was assessed visually. Pre- and post-filtration samples weremixed with a fixative (0.1% glutardehyde in 3.4 Na citrate) toimmobilize the sperm cells. Sperm concentration was determined using ahemacytometer. Total number of sperm cells was calculated on the basisof volume multiplied by the concentration of sperm cells. The recoveryrate was calculated as the total number of sperm cells in the flushedback portion divided by the total number of sperm cells in the aliquotprior to filtration. The process was repeated with a different filter.The experiment tested both of the following filters: (1) a 1.0, μm PTFE(not FTPE) membrane disc (syringe) filter (which is available from PallCorporation, Life Science Group, Ann Arbor, Mich., Cat #PN4226T or VWR,Batavia, Ill., Cat. #28143-928); and (2) 0.8 SFCA (surfactant freecellulose acetate) membrane disc (syringe) filter (Corning, Inc.,Corning, N.Y., Cat. #431221; VWR Batavia, Ill., Cat. #28200-028). Theresults are shown in FIG. 101. More spermatozoa were recovered withcellulose acetate filters as compared to PTFE filter, i.e. 67 vs 33% dueto low protein binding affinity of cellulose acetate. Visual motility ofspermatozoa recovered ranged from 63% (PTFE) to 68% (Cellulose acetate).

Filtration Example II

Filtration example II shows the recovery rate and motility of spermcells after concentration by at two-step filtration process of thepresent invention. Semen samples were collected from three bulls andevaluated as provided in the sample preparation section above. The threesamples were diluted with a quantity of TCA (pH 7.3) necessary to attaina concentration of 150×10⁶ sperm cells/ml. One and one half ml of PBSwith 10% egg yolk and 28.3 ml PBS with 0.1% BSA was added to each of 50test tubes. Two hundred μl of semen sample (about 30×10⁶ sperm cells)was added to each test tube and incubated for twenty minutes at roomtemperature. Referring to FIG. 100, a syringe 2431 was used to applynegative pressure to draw a 6 ml aliquot of the diluted semen 2433 fromeach test tube through a filter 2435. The filter was placed in a filterholder 2437 (a Swinnex filter holder from Millipore Corporation,Billerica, Mass. Cat #SX0002500,). After filtration, the filtrationholder 2437 was disconnected from the syringe and the tubing, keepingthe filter holder intact. Spermatozoa on the filter were collected byturning the filter assembly upside down and back flushing 1 with ml ofTCA buffer using a 3 ml syringe having a small piece of tubing at thetip in a 15 ml test tube. Sperm motility was assessed visually. Pre- andpost-filtration samples were mixed with a fixative (0.1% glutardehyde in3.4 Na citrate) to immobilize the sperm cells. Sperm concentration wasdetermined using a hemacytometer. Total number of sperm cells and therecovery rate were calculated as specified in Filtration example 1. Theprocess was repeated twice to test different filters. The experimenttested both of the following filters: (1) a 0.2 μm Teflon membranefilter (which is available from X-Partek, P. J Cobert Associates Inc.St. Louis Cat. #944106; and (2) a 0.8 cellulose acetate membrane filter(Millipore Corporation, Billerica, Mass. Cat. #AAWP 02500). The resultsare shown in FIG. 102. In both filters, the recovery rate of spermatozoawas low (˜25%). It was low in Teflon filter as in example 1. However,low recovery rate and visual motility of flushed back spermatozoa incellulose acetate filter might be due to the material used by differentvendor and/or ability of spermatozoa to attach with filterholder/assembly.

D. Dense Medium Concentration

Another alternative method of concentrating the collected sperm relieson flotation of sperm cells in a high-density medium. According to thismethod, a high-density medium is added to the collected sperm cells toraise the specific gravity of the suspension above about 1.3. Forexample, a colloidal silica suspension such as is available under thePercoll® and Isolate® tradenames may be used to increase the specificgravity of the suspension. The sperm cells will float to the top of thesuspension, where they can be skimmed or otherwise collected, because ofthe increased specific gravity of the suspension. A resuspension fluidis added to the cells that have been collected from the surface to bringthe final concentration to about 20×10⁶ sperm cells/ml. Some of thesuspension fluid may be removed by one of the filtration methodsdescribed above prior to addition of the high density medium to reducethe quantity of high density medium required to attain the desiredspecific gravity.

Cryoextension

A. Cryoprotection

Once the sperm have been sorted and collected in the collection vessels,they may be used for inseminating female mammals. This can occur almostimmediately, requiring little additional treatment of the sperm.Likewise, the sperm may also be cooled or frozen for use at a laterdate. In such instances, the sperm may benefit from additional treatmentto minimize the impact upon viability or post-thaw motility as a resultof cooling and freezing.

Generally, a cryoextender comprises a buffer or buffered solution, aprotein source, and a cryoprotectant. Examples of buffers and bufferedsolutions that may be used in the cryoextender are disclosed above withrespect to sample collection and extension. Typically, these bufferswill be in a concentration of about 0.001M to about 1.0M and have a pHof about 4.5 to about 8.5, preferably of about 7.0.

If included, a protein source may be added to provide support to thecells and to cushion the contact of the cells with the collectionvessel. The protein source may be any protein source that does notinterfere with the viability of the sperm cells and is compatible withthe particular buffer or buffered solution being used. Examples ofcommon protein sources include milk (including heat homogenized andskim), milk extract, egg yolk, egg yolk extract, soy protein and soyprotein extract. Such proteins may be found in a concentration fromabout 10% (v/v) to about 30% (v/v), preferably from about 10% (v/v) toabout 20% (v/v), and more preferably about 20% (v/v). While milk may beused in combination with a buffer or buffered solution, generally milkis used in the absence of the same, as milk is a solution itself thatmay serve the same purpose of a buffer or buffered solution. In suchinstances, the cryoextender would contain about 80% (v/v) to about 90%(v/v) milk.

A cryoprotectant is preferably included in the cryoextender to lessen orprevent cold shock or to maintain fertility of the sperm. Numerouscryoprotectants are known in the art. Selection of a cryoprotectantsuitable for use with a given extender may vary, and depends upon thespecies from which the sperm to be frozen were obtained. Examples ofsuitable cryoprotectants include, for example, glycerol, dimethylsulfoxide, ethylene glycol, propylene glycol, trehalose, Triladyl® andcombinations thereof. If included, generally, these cryoprotectants arepresent in the cryoextender in an amount of about 1% (v/v) to about 15%(v/v), preferably in an amount of about 5% (v/v) to about 10% (v/v),more preferably in an amount of about 7% (v/v), and most preferably inan amount of about 6% (v/v).

In one particular embodiment, the cryoextender comprises water,Triladyl®, egg yolk, and pyruvic acid. In yet another embodiment, thecryoextender comprises 25 g Triladyl® 25 g egg yolk, and 10 mM pyruvicacid in 75 mL of water.

Optionally, the cryoextender may also contain a range of additives thatare beneficial to sperm viability or motility and that prevent or lessenthe detrimental side effects of cryopreservation. Such additives mayinclude, for example, an energy source, an antibiotic, or a compositionwhich regulates oxidation/reduction reactions intracellularly and/orextracellularly, each of which is discussed above with respect to samplecollection and dilution. Such additives may be added to the cryoextenderin accordance therewith.

B. Cryopreservation of Sorted Sperm Cells

In most cases, it will not be possible to use the sperm cells that havebeen sorted as described above for immediate artificial insemination.Particularly in the case of a commercial sperm sorting operation, thesorted sperm cells must be stored and/or transported before they can beused for artificial insemination. This will usually requirecryopreservation of the sperm cells. The sorted sperm may be loaded intoelongate cylinders (known as “straws” in the breeding industry) andcryopreserved to preserve the sperm during transportation and storage.Cryopreserved sperm cells can be stored for long periods of time inliquid nitrogen. To use the cryopreserved sperm, the straw may beimmersed in a heated water bath to thaw the sperm. Then the straw isloaded into an artificial insemination gun which is used to inseminate afemale animal. Several precautions must be taken to protect the spermcells during cryopreservation. Otherwise the sperm cells will be sodamaged (as indicated by a low post-thaw motility rate of 5-10%) thatthey are not suitable for use in artificial insemination.

Conventional cryopreservation methods involve sequentially adding aprotein source (e.g., egg yolk), cooling the sperm to a temperature ofabout 4-5° C., adding a cryoprotectant (e.g., glycerol), maintaining thesperm and cryoprotectant at a steady temperature in the range of about4-5° C. for a period of time sufficient to allow the sperm cells toequilibrate with the cryoprotectant, and then supercooling the sperm, asby immersing the sperm cells in liquid nitrogen at −196° C. for storage.Those skilled in the art will recognize that the purpose of the proteinsource is to protect sperm from damage as they cool from about 14° C. toabout 8° C., which is the temperature at which sperm cells are mostsusceptible to cold shock. In contrast, the cryoprotectant protects thesperm cells from damage at temperatures below 0° C. Even though thetemperatures involved in cryopreservation are well below freezing andthe term “freezing” is sometimes used to describe cryopreservation,those skilled in the art will also know that cryopreserved sperm are notactually frozen. To be precise, the cryopreserved sperm are in asupercooled state. The conventional period during which sperm cells andcryoprotectant are maintained at a steady temperature can last anywherefrom 60 minutes to many hours. The overall time to completecryopreservation using conventional methods generally exceeds fourhours. Furthermore, it is believed that up to 50% of the sperm cells arekilled in conventional cryopreservation processes. Though sperm arecryopreserved using conventional methods according to some embodimentsof the present invention, other embodiments of the present inventionemploy improved cryopreservation methods to reduce the time required forcryopreservation and/or to improve the health of the cryopreservedsperm.

FIG. 103 shows a work flow diagram outlining the steps of one exemplaryembodiment of an improved method of cryopreserving sperm according tothe present invention. At step 2501, the concentration of a solutioncontaining sorted sperm cells is adjusted to be in the range of about 1million-40 million sperm/ml, depending on the standard used by thetargeted consumer (e.g., breeding association). For example, the spermconcentration may be adjusted to be in the range of about 20 million to24 million sperm/ml. Adjustment of the sperm concentration may includeaddition of resuspension fluid, buffers and/or extenders to concentratedsperm as described above. At step 2503, a cryoprotectant (e.g.,glycerol) is added before the sperm are cooled. The sperm cells beginequilibrating with the cryoprotectant as soon as they come into contactwith the cryoprotectant. At step 2505, a protein source (e.g., egg yolk)is also added to the solution containing the sperm cells as describedabove.

The sperm cell solution, protein source, and cryoprotectant are loadedinto conventional 0.5 or 0.25 ml artificial insemination straws using aconventional loading machine at step 2507. Those skilled in the art willbe familiar with a number of conventional apparatus and techniques thatmay be used to load semen into straws. For example, U.S. Pat. No.5,249,610, issued Oct. 5,1993 to Cassou, et al. and incorporated hereinby reference, provides instruction about the filling of straws withbovine semen using a disposable injector nozzle. Moreover, equipment forfilling straws is commercially available from Minitube of America,located in Verona Wis. Any of these or similar conventional loadingmethods and apparatus can be used to load the sorted sperm cells intostraws.

After loading, the sperm cells are cooled to a holding temperature atstep 2509. In general, the holding temperature should be selected withthe following considerations in mind: holding sperm cells at atemperature that is too high (e.g., 10° C.) may cause unnecessary damagefrom cold shock; equilibration of sperm cells with a cryoprotectant(e.g., glycerol) is believed to be most active at temperatures in therange of 4−5° C.; and holding sperm cells at temperatures that are toolow (e.g., <0°) is believed to be damaging to the sperm cells. Thus,according to one embodiment, the holding temperature is in the range of0−8° C. More desirably, the holding temperature is in the range of 2−6°C. Even more desirably, the holding temperature is in the range of 4−5°C. In another embodiment, the cooling rate used for this step 2509 isselected to minimize damage to the sperm cells. For example, the coolingrate may be controlled (e.g., substantially constant) to providehomogenous cooling and to prevent the sperm from suffering temperatureshock. The cooling rate should also cool the sperm quickly enough toreduce their metabolism before they incur membrane damage, but slowlyenough that they do not suffer from temperature shock. One can controlthe cooling rate by placing the straws containing the sperm cells in aprogrammable freezer (e.g., an IceCube 1810CD freezer which is availablecommercially from Minitube of America, located in Verona, Wis.) to coolthem. According to one embodiment, the programmable freezer cools thesperm from about room temperature (typically in the range of about 22and 24° C.) at a constant cooling rate of 0.1 and 0.3° C./minute. Moredesirably, the cooling rate is in a range of about 0.15 and 0.25°C./min. Even more desirably, the cooling rate is about 0.2° C./min. Inanother embodiment, the cooling rate is selected so the sperm are cooledfrom their initial temperature to the holding temperature in about 90minutes. In still another embodiment, the cooling rate is selected tocool the sperm from their initial temperature to the holding temperatureat a constant cooling rate in about 90 minutes. The cooling ratesreferred to above actually refer to the rate of the cooling of thechamber of the programmable freezer, but because of the thin walls andlong, thin shape of the straw (e.g., about 5.25 inches long, less than 3mm in diameter, and about 0.15 mm in wall thickness) and the conductiveproperties of the straw, the temperature difference between the spermcells and the cooling chamber is not significant.

After the sperm cells have been cooled to the holding temperature, atstep 2511 they are kept at or near that temperature for a period toallow substantial completion of their equilibration with thecryoprotectant. For example, the programmable freezer described abovecan be programmed to hold the sperm cells at a steady temperature duringthe period. According to another embodiment of the present invention,the sperm cells are held at the holding temperature for a period that isshortened compared to conventional methods because the sperm havealready been equilibrating with the cryoprotectant during the coolingprocess. For example, the period may be in the range of about 10 and 60minutes. More desirably, the period is in the range of about 20 and 40minutes. Even more desirably, the period is about 30 minutes. In anotherembodiment the period is less than 60 minutes. In yet anotherembodiment, the period is less than 40 minutes. The relatively shortholding period offers a number of advantages in a commercial spermsorting process. First, it reduces the time required to process sortedsperm which can translate to cost savings. Also, the sperm cells stillperform metabolic processes at temperatures in the range of 0-8° C. soreducing the time for which sperm need to be held at this temperaturecan improve the health of the sperm cells, which will increase the valueof the sperm cells to animal breeders who are concerned about artificialinsemination success rates.

After the sperm cells have been held at the holding temperature for aperiod described above, the sperm cells are cooled at step 2513 to atemperature that approaches the critical temperature zone for spermcryopreservation. Those skilled in the art will know that the criticaltemperature zone is the zone at which ice crystal formation and changesin osmotic pressure damage the sperm cells. This temperature may varydepending on the solution in which the sperm cells are cryopreserved,but the critical temperature zone is generally in the range of −18 and−35° C. Sometimes this critical temperature zone is reported to be inthe range of about −18 and −30° C. Thus, according to yet anotherembodiment of the present invention, the cooling rate used to cool thesperm cells from the holding temperature to a temperature thatapproaches −18° C. (e.g., −15° C.) is selected to protect the health ofthe sperm. Relevant factors to consider include that fact that the spermcells are still equilibrating with the cryoprotectant during thisperiod, the fact that sperm are still performing some metabolicfunctions, and the fact that the sperm are still somewhat sensitive torapid temperature change. Again, it is desirable that the cooling ratebe a controlled rate, such as a rate that may be programmed into theprogrammable freezer described above. More desirably, the cooling rateused to cool the sperm from the holding temperature to a temperaturethat approaches about −18° C. is a constant cooling rate. Thus,according to another embodiment of the present invention, the spermcells are cooled from the holding temperature to about −15° C. at acooling rate in the range of about 1.0-5.0° C./min. More desirably, thecooling rate is in the range of about 2.0-4.0° C./min. Even moredesirably, the cooling rate is about 3.0° C./min.

Step 2515 involves rapidly cooling the sperm cells through the criticaltemperature zone to limit the time sperm cells dwell therein. Thus,according to one embodiment of the present invention, the cooling ratethrough the critical temperature zone about (e.g., −18° C. to about −30°C.) is selected to be much faster than the cooling rate used to coolsperm cells to the holding temperature and the cooling rate used to coolsperm cells to the temperature approaching the critical temperaturezone. Thus, the steeper cooling rate is desirably in the range of fromabout 8-40° C. per minute. More desirably, the steeper cooling rate isin the range of from about 8-12° C. per minute. Most desirably, thesteeper cooling rate is about 10° C. per minute. The temperature rangeover which the steeper cooling rate is used may extend beyond thecritical temperature zone. Thus, in yet another embodiment of thepresent invention, the sperm cells are cooled at one of the steepercooling rates described above from about −15° C. to about −40° C. Instill another embodiment, the sperm cells are cooled at one of thesteeper cooling rates described above from about −15° C. to about −80°C. The step of cooling the sperm through the critical temperature zoneat a steeper rate may be accomplished in the programmable freezerdescribed above.

After the sperm cells have been cooled below the critical temperaturezone (e.g., to −80° C.), the straws containing the sorted sperm areimmersed in liquid nitrogen (−196° C.) at step 2517 to provide maximumuseful life of the sorted sperm cells. The use of liquid nitrogen tostore cryopreserved sperm is widespread in the animal breeding industryin the context of unsorted sperm. Thus, those skilled in the art will befamiliar with technologies involving the transportation and storage ofsperm in liquid nitrogen, which need not be discussed in great detailherein. It is sufficient to note that conventional containers areavailable to provide for long term storage of bulk quantities ofartificial insemination straws in liquid nitrogen and that smaller andmore portable containers are also available for providing storage ofartificial insemination straws in liquid nitrogen for transport tocustomers and/or for transport to a farm having one or more femaleanimals to be inseminated with cryopreserved sperm.

One advantage of the cryopreservation methods described herein is thatthe cryopreservation can be completed in less time than is requiredaccording to conventional methods. Perhaps relatedly, the decline inmotility due to cryopreservation according to the present invention isonly about 5-11%, as indicated by the example discussed below. Thus,cryopreservation according to the present invention preserves the healthof the sperm cells as indicated by tests showing that sperm cellscryopreserved according to the present invention have greater than 50%(e.g., about 60%) motility after they are thawed in a 37° C. water bathfor about 50 seconds. As discussed above, sperm motility may be analyzedby an automatic machine (e.g., the IVOS sperm analyzer from HamiltonThorn Research) or by visual examination.

It should be noted that the cryopreservation methods described above arecontemplated as being used in a commercial scale sperm sorting process.Thus, according to one embodiment of the present invention, the steps ofthe inventive methods described herein are performed simultaneously on abatch of sorted sperm cells to quickly cryopreserve the entire batch ofsperm cells in a manner that preserves their health. For example, byusing the multi-channel flow cytometry apparatus described below, it ispossible to obtain about 840×10⁶ sorted X chromosome-bearing sperm cellsin the collection system of the apparatus in about 20 minutes. This isenough sperm cells to fill several dozen straws. Moreover, a batch caninclude the combined sperm cells by two or more different sortingcytometers. After being concentrated as described above, the sperm cellscan be loaded into any number of straws and cryopreserved as a batch.For example, according to one embodiment of the invention, it takesabout 5 minutes to add an extender (including both a protein source anda cryoprotectant) to a batch of sperm cells, and about 15 minutes toload the sperm cells into artificial insemination straws using anautomatic loading machine. All the straws in the batch are cooledsimultaneously in a programmable freezer. Furthermore, the capacity ofsome programmable freezers allows simultaneous cryopreservation ofthousands of artificial insemination straws. For example, the IceCube1810CD freezer referred to above has the capacity to cryopreservesimultaneously over 2,5000.5 ml straws or over 3,800 0.25 ml straws.Thus, one could wait to start the cooling step until multiple batcheshave been obtained. Alternatively, multiple batches could be obtainedsubstantially at the same time by running multiple multi-channel flowcytometry machines (see below) in parallel and simultaneously coolingmultiple batches obtained therefrom together in a programmable freezer.In one embodiment of the present invention, it takes a period of lessthan 220 minutes to cool the sperm cells from room temperature to asupercooled state and immerse them in liquid nitrogen (−196° C.). Inanother embodiment, the supercooling period is less than 190 minutes. Instill another embodiment, the supercooling period is less than 150minutes.

Those skilled in the art will recognize that substantial modificationsmay be made to the foregoing exemplary methods without departing fromthe scope of the present invention. For example, the sperm cells may becryopreserved in a container other than an artificial inseminationstraw. Likewise, the steps in the method that involve changing ormaintaining temperature may be performed by any suitable means,including water baths, liquid nitrogen vapors, and conventionalprogrammable or non-programmable freezers, for example. Furthermore, awide variety of substances or combinations of substances could be usedas the protein source and/or the cryoprotectant without departing fromthe scope of the present invention. These substances include substancesand concentrations of substances listed above in connection with thediscussions regarding buffers, extenders, cryoprotectants, sheathfluids, and collection fluids. Moreover, the order of some steps in themethod may be varied without departing from the scope of this invention.Although FIG. 95 indicates that the cryoprotectant is added after theconcentration of the sorted sperm is adjusted, it is also contemplatedthat a cryoprotectant can be added before the concentration is adjustedwithout departing from the scope of the present invention. For example,the cryoprotectant may be provided in the collection fluid or in thesheath fluid used in connection with a flow cytometer. Some of thebenefits of the present invention may also be obtained by partiallycooling the sperm cells and then adding the cryoprotectant. Likewise,the order in which the protein source is added may be varied as long asthe protein source is effective to protect the sperm cells from coldshock as they pass through the temperature range of about 14 to 8° C.

Cryopreservation Example I

Bovine semen was collected, transported, and evaluated as describedabove. Two test tubes containing 5 ml each of TCA buffer (pH 7.3) wereplaced in one of two water baths for at least five minutes. One waterbath was at a temperature of 35° C. and the other water bath was at 41°C. Spermatozoa at 24° C. were added to each tube so that the finalconcentration in each tube was 150×10⁶ sperm/ml. The two tubes were eachdivided into two aliquots which were kept in respective water baths.After the sperm had equilibrated with the TCA buffer for five minutes,80 μM Hoechst 33342 was added to one of 35° C. aliquots and one of the41° C. aliquots. After addition of the Hoechst 33342, all four aliquotswere incubated for 20 minutes in their respective water bath. Afterincubation, the test tubes were removed from the water baths and left atroom temperature (about 25° C.) for five minutes. Then the contents ofeach test tube were diluted with a TCA extender containing 20% egg yolkand 6% glycerol (v/v) (pH 7.0) to a final concentration of 20×10⁶sperm/ml. The contents of each test tube were then used to fill a 0.5 mlartificial insemination straw. Each of the four straws was placed in aprogrammable freezer (an IceCube 1810CD freezer from Minitube ofAmerica, Wis.). The following cooling sequence was programmed into theprogrammable freezer: (1) 22° C. to 4° C. @-0.2° C./min; (2) hold at 4°C. for 30 min; (3) 4° C. to −15° C. @-3.0° C./min; and (4) −15° C. to−80° C. @-10.0° C./min. After reaching −80° C., the straws were immersedin liquid nitrogen (−196° C.) for 45 minutes. Then the straws wereimmersed in a 37° C. water bath for 50 seconds to thaw. Sperm motilitywas checked under a phase contrast microscope both before and aftercryopreservation. The results are shown in FIG. 104. The post-thawmotility was generally on the order of 60%. This represents a decline inmotility of only about 5-11% compared to before cryopreservation.Analysis of variance revealed no significant effect of either Hoechst33342 or incubation at 41° C. on post-thaw sperm motility.

Operation of the System

The overall operation 813 of the flow cytometry system 9 will now bedescribed with reference to FIG. 82 and in the specific context of spermcells (e.g., bovine sperm cells), but it will be understood that thedescription is exemplary only, and that the system can be used toprocess other types of particles.

The first series of steps leading up to the six second repeat loopinvolve calibration of the system. After initializing 769, a systemcheck 771 is performed to confirm, among other things, that theprocessor 131 or processors are operational. If an error is detectedafter three failed system checks 775, user interaction 773 is requested.If the system check is positive, the microprocessor directs the systemto flush 777 the nozzle system with a suitable fluid, and then a qualitycontrol material 779, such as beads or bovine nuclei, are run throughthe system to initialize the detection parameters (see 739 in FIG. 72)and confirm that the system is operating within an acceptable qualitycontrol. This involves an evaluation of the control material to test thesensitivity and precision of the system to confirm that the system canadequately discriminate a sample. If the quality control is notconfirmed after three attempts 775, user intervention 773 is requested.

If the quality control material indicates an acceptable level of qualitycontrol, a sample 781 is aspirated and a portion or aliquot of thesample to be sorted is checked for quality 783. Sample quality may bedetermined by a calculation of a quality factor (Q-factor) of thesample. For example, the type of cells may be detected in a firstaliquot of the sample. During this detection, the initialized detectionparameters (741) are rechecked and the initial discrimination parameters(745) are generated. If the type of cells detected in the aliquotindicates that the sample meets or exceeds a preset standard (e.g., thatthe sample can be discriminated to yield a certain purity or motilityand, in particular, that there are sufficient live X cells available forprocessing), then the system continues operation. If sample qualityfails three times 775, user interaction is requested.

Continued operation involves sorting 785 of the remainder of the sampleemploying a six second repeated loop. At the beginning of the loop, themicroprocessor confirms that sorting of the sample is not complete 789.If the sorting of the sample is complete 789, the microprocessorproceeds to aspirate the next sample 781 if it is available or to turnoff the sorting operation 793 if additional sample is not available. Ifthe sample is not complete 789, the microprocessor initially checks theX/Y discrimination 795 of the sample to confirm that it is within anoptimum range. In other words, drift analysis as noted above (761 inFIG. 72) is conducted. If any changes should be made, such changes areimplemented and the discrimination 795 is again checked. If thediscrimination is still unacceptable at this point, the sort is turnedoff 793 and user interaction is requested.

Otherwise, the system proceeds to determine whether the fluid deliverysystem is delivering fluid and cells at a rate which is within anoptimum range 801. This determination depends on the type of controlstrategy used. For the high recovery control strategy, the optimum ratewould be determined by evaluating purity or looking at x/x+˜X of thecollected population. If the determined purity is higher than a requiredpurity level, the feed input rate of the cells is increased byincreasing a rate control signal provided to the syringe pump 803. Thiswould tend to increase coincident cells and decrease purity because morecoincident cells including ˜X cells would be collected with the X cells.If the determined purity is lower than the required purity, the feedinput rate of the cells is decreased by decreasing a rate control signalprovided to the syringe pump to reduce the frequency of coincident cells803. Thus, the cell input rate is a function of the determined purity ofthe collected population as compared to a desired purity level, e.g., afunction of the identified ˜X sperm cells collected.

For the high purity control strategy, the optimum rate would bedetermined by calculating lost X cells, e.g., discarded X/discardedX+collected X. If the quantity or percentage of lost X cells are lessthan an acceptable level, the input rate of the cells is increased byincreasing a rate control signal provided to the syringe pump 803. Thiswould tend to increase coincident cells and increase the number ofdiscarded X cells because more cells including X cells would bediscarded with the Y cells. If the quantity or percentage of lost Xcells is higher than the acceptable level, the input rate of the cellsis decreased by decreasing a rate control signal provided to the syringepump 803 to decrease coincident cells. Thus, the cell input rate is afunction of the determined lost X cells of the discarded population ascompared to number of X cells in the collected population, e.g., afunction of the number of X sperm cells not collected.

If this modified rate is acceptable 805, the system proceeds to anothersystem check 807. If the system check is acceptable 807, the sortcontinues in the six second loop. If not, the system is reset 809. Ifafter reset the system is not acceptable or if the revised feed rate isnot acceptable 811, the sort is turned off 793 and user intervention isrequested 773.

The sorted droplet streams are collected by the collection system 2201.Droplets that are sorted into the population of X cells pass through theexit window 2245 in the first intercepting device 2247 to be interceptedby the second intercepting device 2249. From there, the dropletscontaining the X cells flow into a collection vessel 2207. Otherdroplets are intercepted by the first intercepting device 2247 anddirected to the waste trough 2805. Of course droplets intercepted by thefirst intercepting device could also be saved, as noted above. When asuitable amount of X-bearing sperm cells have been collected in thecollection vessel, sorting may be interrupted to allow concentration ofsperm cells in the collection vessel 2207. A new collection vessel maybe placed under the first intercepting device 2247 or the collectedfluid may be poured into a different container and the collection vesselreplaced. Then sorting may resume. The sperm cells in the collectedfluid are concentrated, loaded in straws, and frozen as described above.

Temperature Control During Operation

Temperature control throughout the process may be used to improve theresults of the process. As has already been discussed above, thetemperature of the sperm may be controlled during various steps in theprocess (e.g., staining and cryopreservation). In several embodiments ofthis invention, the temperatures of the sperm cells throughout thevarious steps of the method are controlled to achieve improved results.

For example, FIG. 105 is a work flow diagram of one embodiment of amethod of temperature control according to the present invention. Thetemperature of semen samples at the time they are collected will bedetermined by the body temperature of the animal from which they arecollected. For example, at step 2601 bovine semen samples are collectedat about 37° C. An insulated container is used for transportation of thesemen samples to the lab from the collection site at step 2603. Theinsulated container retards cooling of the sperm.

During sample evaluation at step 2605, the temperature is maintainedbelow the collection temperature, but in excess of a temperaturecorresponding to a glass transition temperature below which the spermcells suffer membrane damage. For example the temperature may bemaintained in the range of about 18-37° C. In another embodiment, thetemperature may be maintained in the range of about 24-37° C. duringsample evaluation. In a particular embodiment, the sperm cells areplaced in an environment having a temperature in the range of about22-25° C. during sample evaluation. Depending on the temperature of thesperm upon arrival at the lab, the effect of placing them in anenvironment having a temperature in the range of about 22-25° C. may beto continue slow cooling of the sperm, to maintain the temperature ofthe sperm, or to slightly raise the temperature of the sperm. In oneembodiment, the temperature may be elevated (e.g., to 40° C. or higher)for staining at step 2607 as discussed in the staining section. Inanother embodiment, the temperature of the sperm cells during thestaining step may be in the range of about 20-40° C., as is alsodiscussed above.

At step 2609, the stained semen mixture is held in a water bath untilsuch time that the mixture is introduced into a flow cytometer. Thetemperature of the water bath may be similar to the temperature used forthe staining step. In one embodiment the temperature of the water bathis in the range of about 40-47° C. In another embodiment, thetemperature of the water bath is in the range of about 20-37° C. Instill another embodiment, the temperature of the water bath is in therange of about 20-25° C. After being held in the water bath for any timebetween one minute and two hours, the stained sperms cells are sorted byflow cytometry as discussed above at step 2611. At step 2613, thecollected sperm cells are concentrated. Concentration may be performedin an environment that has a temperature that will not significantlychange the temperature of the sperm cells. For example, in oneembodiment, concentration may be performed in an environment having atemperature in the range of about 20 and 25° C. An extender, proteinsource, and cryoprotectant are added to the concentrated sperm at step2615. Then, at step 2617 the sperm cells are loaded into artificialinsemination straws. In one embodiment, the loading step is performed inan environment having a temperature that will not significantly changethe temperature of the sperm cells. Finally, at step 2619 thetemperature of the sperm is controlled during cryopreservation asdiscussed above.

In another embodiment, sperm cells may be stained at still lowertemperatures without departing from the scope of the present invention.For example, it may be desired to sort the sperm cells in a flowcytometer at a relatively low temperature (e.g., about 0° C. to about 8°C.). This may require modification of the overall temperature control.First, when cooling the sperm cells prior to introduction into a flowcytometer, egg yolk and other common protein sources that protect thesperm cells from cold shock at temperatures below the glass transitiontemperature generally may not be used as such protein-containingsubstances tend to foul and/or clog the fluidics of the flow cytometer.Thus, it is desirable to cool the sperm cells before performing thestaining step in order to take advantage of natural cold shockprotectants found in neat semen, such as for example, the seminal fluid.Any attempt to stain the sperm cells prior to cooling would requireaddition of buffers to protect the sperm which would dilute the neatsemen and reduce the natural protection against cold shock.

Accordingly, one embodiment of the present invention for sorting thesperm cells at a temperature in the range of about 0° C. to about 8° C.includes placing the sperm cells in an environment having a temperatureless than about 8° C. to cool the sperm cells to a temperature in therange of about 0° C. to about 8° C. prior to staining. Any method may beused to cool the sperm cells, but it is desirable to use a method thatprotects against rapid temperature fluctuations of the sperm cellsduring the cooling process. For example, in one embodiment, a containerholding the sperm cells is placed in a room temperature water bath,which in turn is placed in an environment having a temperature less thanabout 8° C. In another embodiment, the temperature of the sperm cells ismonitored and ice is added to the water bath to further cool the spermcells. The staining step may be performed as described above except thatthe staining mixture is subjected to a temperature in the range of about0° C. to about 8° C. Due to the lower temperature, the incubation periodrequired to stain the cells is considerably longer. Once the sperm cellshave been cooled to 8° C. or below, it is desirable to avoid warmingthem. Thus, another embodiment of the present invention is to operatethe flow cytometer in an environment having a temperature in the rangeof about 0° C. to about 8° C. Similarly, another embodiment of thepresent invention is to collect the sorted sperm cells in a collectionvessel that is surrounded by an environment having a temperature in therange of about 0° C. to about 8° C. Still another embodiment of thepresent invention is to add any extenders, cryoprotectants, buffers,protein sources, antibiotics, antioxidants, or other additives at atemperature in the range of about 0° C. to about 8° C. With respect toaddition of the cryoprotectant, it may be desirable to add slightly moreof the cryoprotectant than would be added absent sorting the sperm cellsat a temperature in the range of about 0° C. to about 8° C. Thus, in oneparticular embodiment, a cryoprotectant containing 7% glycerol (v/v) isadded to sperm cells after the sperm cells have been sorted at atemperature in the range of about 0° C. to about 8° C.

Supercooling of the sperm cells from the temperature in the range ofabout 0° C. to about 8° C. proceeds generally as described in thecryopreservation section above. However, the sperm cells will need to beheld at a temperature in the range of about 0° C. to about 8° C. for aperiod of time after addition of the cryoprotectant before supercoolingto allow time for the sperm cells to equilibrate with thecryoprotectant. Thus, according to one embodiment, the sperm cells areallowed to equilibrate with the cryoprotectant for a period in the rangeof about 30 minutes to about 3 hours. In another embodiment, the spermcells are allowed to equilibrate with the cryoprotectant for a period inthe range of 1-2 hours. In another particular embodiment, the spermcells are allowed to equilibrate with the cryoprotectant for a period ofabout 90 minutes.

Conventional temperature control apparatus and methods (e.g., waterbaths, incubators, coolers, and freezers) may be used to heat or coolthe sample to attain or maintain the specified temperatures in theforegoing embodiments of the invention. It is understood that placing asample in an environment having a different temperature than the sample,will cause the temperature of the sample to change over time. There mayeven be temperature variations within the sample. As has been mentioned,it is desirable to change the temperature of the sample gradually tohelp maintain the health of the sperm. Gradual temperature change alsoserves to reduce the temperature variation within the sample. As is wellknown by those skilled in the art, the rate of temperature change of thesample will be influenced by many factors, including the volume of thesample, the size and shape of the sample container, and the magnitude ofthe temperature difference between the sample and the environment.However, those skilled in the art will readily be able to select anappropriate method and apparatus to achieve the desired temperaturecontrol after considering all the relevant factors.

Those skilled in the art will recognize that there is room forsubstantial variation in the exemplary temperature control withoutdeparting from the scope of the invention. In general, once the spermcells have been chilled, it is desirable to avoid warming them.Furthermore, temperature variations discussed above in connection withsample collection, staining, sorting, droplet collection, concentration,and cryopreservation can be incorporated into the overall temperaturecontrol without departing from the scope of the present invention.Moreover, the time at which sperm cells remain at any temperature canalso impact the health of the sperm. Thus, processing according to theembodiment in which temperature is controlled throughout the process isdesirably completed within a timeline as discussed below.

Timeline for Operation

Generally, it is desirable to complete the sperm sorting process in theleast amount of time possible to reduce the damage to the sperm. Asdiscussed above, the present invention may include staining at anelevated temperature to reduce the time needed to stain the sperm cells.For example, certain embodiments of the improved staining methoddescribed reduce the time require for staining to about 10 minutes.Likewise, the novel cytometer described above may be used to sort spermcells in less time than would be required by a conventional cytometer.For example, a flow cytometer using the technology discussed above cancollect between 2,000 and 10,000 sperm cells having a desired DNAcharacteristic per second. Furthermore, the cryopreservation process maybe used to reduce the time needed to complete cryopreservation of theprocessed sperm cells compared to conventional cryopreservation methods.Accordingly, one embodiment of the present invention involves processingsperm pursuant to an overall method to take advantage of one or more ofthe timesaving innovations to reduce the time required to complete theentire process. For example, according to one embodiment of the presentinvention, a batch of sperm cells (e.g., an ejaculate) is collected froma male mammal (e.g., bull), evaluated for quality control, stained,sorted according to a specified DNA characteristic, loaded into one ormore containers (e.g., straws), and cryopreserved within a period ofabout 12 hours from the time of collection. In another embodiment, theperiod is less than about 8 hours. In another embodiment, the period isless than about 6 hours. In still another embodiment, the period is lessthan about 3 hours. In yet another embodiment, the period of time isless than about 2 hours. In another embodiment, the period of time isless than about 1 hour.

Multi-Channel Sorting Apparatus and Method

In order to sort more sperm in less time, it is possible to use morethan one cytometry unit in parallel to sort that same sperm sample. Oneway to do this is to simply divide the stained sperm cells into multiplealiquots and run each aliquot through a different cytometer. However, aswill be discussed below, certain advantages may be obtained by designingan apparatus that comprises multiple cytometry units in a singleintegrated multi-channel cytometry unit.

Multi-Channel System Sharing Integrated Platform

FIGS. 106-116 show one embodiment of the invention comprising amulti-channel cytometry system, generally designated 1001, wheremultiple single-channel flow cytometry units, designated 1003, areganged together as an integrated system to produce sorted product. Foursuch units are illustrated in this particular embodiment, but thisnumber can vary. The units may be integrated in various ways, as bysharing an integrated platform comprising one or more of the followingelements (1) a common supply of particles 1005; (2) a common source ofelectromagnetic radiation 1007; (3) a common housing 1009; (4) a commoninput for controlling operation of the units 1011; (5) a common output1019 allowing evaluation of the operation of one unit relative toanother unit; (6) a common fluid delivery system 1021; (7) a commontemperature control system 1023; (7) a common power source 1025; (8) acommon waste recovery system 1027; (9) a common deflector plate system1029; and (9) a common cleaning system 1031. In one embodiment, thesystem includes all of these elements, but it will be understood that amulti-channel system of this invention can include any combination ofelements. The use of common elements is beneficial because it allows thesystem to be run more efficiently and profitably, achieves moreconsistent results among channels by reducing the number of variables,facilitates any trouble-shooting that may be needed, and is economical.The multi-channel approach also makes the sorting system more amenableto scale up or scale-down.

Each of the cytometry units 1003 has components similar to certaincomponents of the flow cytometry apparatus 9 of the previous embodimentand, for convenience, corresponding parts are designated by the samereference numbers with the addition of a prime (′). In general, eachunit comprises a nozzle system 101′, a mount for mounting the nozzlesystem 331′, a transducer 105′, and an epi-illumination opticsinstrument 417′ for focusing a beam of light 25′ on the fluid stream 21′exiting the nozzle orifice 103′, all as previously described. Each unitfurther comprises a photodetector 117′ operable as in the firstembodiment to detect fluorescence emissions 31′ from the particles inthe stream 21′ and to convert the emissions 31′ to electrical signals701′ which are processed to classify the particles by a specified DNAcharacteristic. Each unit 1003 is also equipped for sorting the droplets33′ into different groups or populations 123′, 125′ according to theclassification of particles contained in the droplets 35′. Thepopulations of droplets sorted by the units are collected by thecollection system 2201.

A. Common Housing and Modularity

The flow cytometry units are mounted in a modular arrangement in acommon housing 1009. In the embodiment shown in FIGS. 106 and 109-113,the housing has a base 1069 and two side walls 1071 extending up fromthe base. The side walls have a lower pair of shoulders 1073 forsupporting a lower cover panel 1075 at the front of the housing 1077,and an upper pair of shoulders 1081. A lower cover panel 1075 at thefront of the housing 1077 is mounted between the lower shoulders 1073.The upper shoulders 1081 support an upper cover panel 1083 at the rearof the housing 1085. The front and rear of the housing 1077, 1085 aresubstantially open to provide access to the equipment inside. It will beunderstood that the housing 1009 may have other configurations withoutdeparting from the scope of the invention. Further, it will beunderstood that the various units could be installed in separatehousings.

The flow cytometry units 1003 are mounted side-by-side as modules on anappropriate framework 1087 in the housing 1009. Specifically, the nozzlemounts 331′ for positioning the nozzles 101′ are releasably attached toa cross bar 1089 (FIG. 106) affixed to the side walls 1071 of thehousing, and the bases 429′ of the epi-illumination instruments 417′ arereleasably fastened to an angled mounting plate 1093 extending betweenthe side walls 1071 of the housing toward the rear of the housing 1085(FIG. 109), the arrangement being such that a particular unit can beinstalled or removed as a module. This modularity facilitatesinstallation, removal for maintenance and/or replacement, and enablesany number of flow cytometry units 1003 to be readily added as needed ordesired to increase the throughput capacity of the system.

B. Common Fluid Supply and Delivery Systems

The fluid delivery system 1021 of this embodiment is equipped to provideappropriate fluids to each of the cytometry units 1003. As illustratedschematically in FIG. 108, the system generally comprises a pump 1105for conveying carrier fluid 17′ from a common supply of carrier fluid1107 under pressure, a gas pressure system 1115 for conveying fluid froma common supply 1117 of sheath fluid 19′ under pressure, and a manifoldsystem 1121 for receiving the fluids from respective supplies anddelivering the fluids under pressure to the various cytometry units1003, as needed. In the specific embodiment of FIG. 116, the supply ofcarrier fluid comprises a vessel 1123 containing a suitable volume ofsuch fluid (e.g., 5 ml.). The vessel is held by a holder 1125, which maybe a block 1133 having a cavity 1135 sized to receive the vessel 1123.The block also has a second cavity 1137 for holding a vessel 1139containing a suitable buffer material for conditioning the system duringuse, as will be described later.

The pump 1105 for delivering carrier fluid from the vessel is desirably(but not necessarily) a syringe pump 1141 as previously described. Theplunger of the pump is movable through an intake stroke to aspirate aselected volume of carrier fluid 17′ from the vessel 1139 and through adischarge stroke to dispense carrier fluid through a supply line 1147 tothe manifold 1177 and from there to the various nozzles 101′ of thesystem. The syringe pump is also operable to aspirate fluid from thevessel 1139 containing buffer and to pump the buffer through the systemin a manner to be described. A three-way valve 1149 controls the flow ofcarrier and buffer fluids to and from the pump 1141. The pump is drivenby a variable speed motor under the control of the processor 131′. Byway of example, the pump may be driven by a stepper motor which operatesat selectively variable speeds to pump carrier fluid to the manifoldsystem 1121 at rates necessary to obtain the desired throughput from theunits 1003. Multiple syringe pumps or other types of fluid deliverydevices can be used instead of a single syringe pump.

In one embodiment the supply 1117 of sheath fluid comprises a vessel1155, e.g., a tank connected to the manifold 1177 by means of a supplyline 1157. The gas pressure system 1115 is operable to pressurize thetank and comprises a source of pressurized gas 1161 (e.g., air ornitrogen) communicating with the tank via a gas line 1163 having aregulator 1165 in it for controlling the pressure supplied to the tank,and a two-way valve 1167 which, in a first position, establishescommunication between the tank and the gas source, and in a secondposition, is operable to vent the tank. The gas pressure regulator 1165is a conventional regulator adjustable to control the pressure suppliedfrom the air source. The gas pressure system 1115 also includes a gasline 1169 for pressurizing a supply 1173 of cleaning solution (e.g.,de-ionized water in a tank) which can be used to flush the fluidcircuitry in a manner to be described hereinafter. Flow through the gasline is controlled by a two-way valve 1167 operable in the same manneras valve 1167.

In one embodiment, the manifold 1177 comprises a laminated block 1179(FIG. 112) of material having passages 1181 formed in it to define afluid flow circuit 1185 such as that shown diagrammatically in FIG. 116.(The passages may be formed by machining grooves in faces of thelaminations prior to assembly of the laminations to form the block.) Thefluid circuit includes inlets 1189, 1191 connected to the syringe pump1141 and to the supply 1117 of sheath fluid, and sets of outlets 1193,for providing such fluids to the flow cytometry units 1003, each suchset including a carrier fluid outlet and a sheath fluid outlet. Flowthrough the various flow passages 1181 is controlled by valves V1-V6which, in one embodiment, are solenoid-operated valves in housingsattached to the manifold block 1179. The block is desirably ofsubstantially transparent material (e.g., acrylic plastic) to facilitatemonitoring of the system 1121 and trouble-shooting. In the embodimentshown, the manifold 1177 is attached to a frame member 1203 extendingbetween the side walls 1071 of the housing 1009 adjacent the bottom ofthe housing below the nozzle systems 101′. The inlets and outlets 1193,of the manifold 1177 may comprise fittings 1205 threaded into the block,such as flangeless nut and ferrule fittings available from UpchurchScientific, a division of Scivex. It will be understood that the designand construction of the fluid circuit 1185 in general and the manifold1177 in particular may vary without departing from the scope of thepresent invention.

Referring to FIG. 116, the manifold fluid circuit 1185 for the carrierfluid 17′ includes a sample reservoir 1207 for holding a limited supplyof carrier fluid (e.g., 1.0 ml). If the carrier fluid contains spermcells, for example, providing such a reservoir close to the nozzles 101′is beneficial to the viability and motility of the sperm cells, sincethe storage of such cells, even for short periods of time, in smallspaces may be detrimental to motility. Flow of carrier fluid from thesample reservoir 1207 to the nozzles 101′ is controlled by a series oftwo-way valves V1A-V1D, one for each nozzle. Each valve V1A-V1D has afirst position establishing fluid communication between the needle 1217of the sample reservoir and the needle 157′ of a respective nozzle fordelivery of carrier fluid 17′ to the needle under pressure generated bythe syringe pump 1141, and a second position establishing fluidcommunication between the needle 1217 and a waste system, generallydesignated 1221, which is common to all of the flow cytometry units1003. In the embodiment shown, the waste system 1221 comprises a wastetank 1223 for holding waste material, a mechanism 1225 such as a vacuumpump for generating a vacuum in the waste tank, and waste lines 1227connecting the valves V1A-V1D and the waste tank. A valve V3 is providedin the waste line upstream from the waste tank for opening and closingthe waste line, as needed. A suitable hydrophobic filter 1233 isprovided in the line connecting the waste tank 1223 and the vacuum pump1225.

The manifold fluid circuit 1185 for the sheath fluid 19′ includes aplurality of valves V2A-V2D. Each valve has a first positionestablishing fluid communication with the supply 1117 of sheath fluid inthe tank for delivery of sheath fluid 19′ to a respective flow body 133′via a sheath supply line 1241, and a second position establishing fluidcommunication between the flow body and the waste tank via a waste line1247. The pressure at which the sheath fluid is delivered to the flowbodies 133′ will depend on the sheath tank pressure (as controlled bythe regulator 1165) which may range from 1 to 100 psi, more desirablyfrom 10 to 50 psi, even more desirably 15 to 40 psi, and even moredesirably from about 20 to 30 psi.

While the use of a common supply for all of the units has variousadvantages, it is contemplated that at least some of the flow cytometryunits could be supplied with sample material from separate sources.

C. Common Power Supply and Input and Output Controls

The flow cytometry units 1003 also share a common power supply 1025,common power delivery systems, a common input (GUI) 715′ for controllingoperation of the channels by the microprocessor 131′, and a commonoutput provided to the microprocessor allowing evaluation of theoperation of one channel relative to another channel. For example, thecommon output includes providing the digitized signals from eachepi-illumination system to the microprocessor for an indication of thefluorescence intensity measured by each channel, for an indication ofthe rate at which each channel is separating particles, for anindication of the staining variations (which may be indicated by theintensity difference of fluorescence pulses from X and Y cells) and foran indication of the decision boundaries 763 used by each channel fordiscriminating between particles. As another example, the common outputincludes providing the output signals from the break-off sensors 389′ tothe microprocessor for an indication of the droplet break-off location107′ of each channel.

D. Common Temperature Control

Optionally, a temperature control system, generally designated 1257, isprovided to regulate the temperature of the contents of the vessels 1123in the holding block 1133 and the temperature of the manifold 1177. Suchtemperature control reduces the variability of the system, thusproviding more consistent measurements between channels and, for certaintypes of cells (e.g., sperm cells), helping to maintain the viability ofthe cells.

In one embodiment, the temperature control system 1257 comprises a fluidflow circuit 1259 comprising fluid passages 1263 in the holding block1133 and fluid passages 1269 in the manifold block 1179, and a controlunit 1265 for circulating a thermal fluid (e.g., water) through thecircuit at a selected temperature. The temperature is desirably such asto maintain the fluid, especially the carrier fluid, at an optimaltemperature to maximize cell viability and, if sperm cells are involved,sperm motility. A valve shut-off V6 is positioned in the circuit forcontrolling flow through the circuit. The temperature control unit maybe used to maintain the sperm cells at the desired temperature prior tosorting as discussed above.

All of the valves in the fluid delivery system 1021 are operated byconventional means, such as solenoids, under control of an operator orsuitable programming. The various fluid flow lines connecting thecomponents of the system outside the manifold block 1179 are desirablyof substantially transparent plastic tubing for observing any blockages.For example, the tubing may be 0.0625 in. OD tubing of FEP polymer. Theflow lines of the temperature control system 1257 are desirably somewhatlarger (e.g., 0.125 in. OD) to provide greater flow capacity.

E. Common Light Beam and Beam Splitting System

As previously noted, the multi-channel system shares a common source ofelectromagnetic radiation or beam light 1007. By way of example (and notlimitation), the source may be a laser beam from a UV multiline laserprimarily having wavelengths of 351.1 nm and 363.8 nm. Alternatively, itmay be desirable to use a pulsed laser (e.g., a mode-locked laser),particularly to synchronize digital sampling with a pulsed laser (asdiscussed in the pulsed laser section) in order to increase theeffective power delivered to each cytometry unit, thereby increasing thenumber of cytometry units that can be operated with a single laser.

The power required to generate the laser beam will vary depending on therequirements of each flow cytometry unit and the number of units. Forexample, if there are N units and each unit requires a light beam havingan effective power of W watts, then it will be necessary to generate alaser beam having a power of (W x N)+L, where L equals the system powerloss among the optical elements of the system. Using a single laser tosupply all of the flow cytometry units is economical compared to asystem using multiple lasers. It is also efficient and provides for moreconsistent measurements from one channel to the next, because there isno variability on account of different beam characteristics (e.g., beamintensity, light polarity, beam divergence) or electrical noiseresulting from the use of multiple lasers.

According to one embodiment of the present invention, a beam splittingand guidance system is used to split a single laser beam into three ormore separate beams. As shown in FIG. 117, for example, a 50/50 beamsplitter 1270 (i.e., a beam splitter that is operable to divide a singlebeam into two separate beams having approximately equal intensity) canbe used to split a single beam 25′ into two beams 1270A, 1270B. By usinga second 50/50 beam splitter 1271 to split one of the two beams 1270Binto two additional beams 1271A, 1271B, one can generate a total ofthree beams 1270A, 1271A, 1271B from a single beam 25′. Each beam can bedirected into the optics system of a flow cytometer, for example anepi-illumination optics system 415′ as shown in FIG. 117. One could alsouse additional 50/50 beam splitters to split the single laser beam intoany number of additional separate beams. As shown schematically in FIG.118, for example, a third beam splitter 1272 can be added to the 3-waybeam splitting system (FIG. 117) so that the three 50/50 beam splitters1270,1271, 1272 can be used to split a single 25′ beam into fourseparate beams 1271A, 1271B, 1272A, 1272B. From this one can readilyappreciate that the single beam can be split into any number of separatebeams. Other beam splitting arrangements may be used to split theincoming source beam into multiple light beams for the various units.

One desirable embodiment of a beam splitting system is shown in FIGS.106 and 109. A beam guidance system 1273 is provided for guiding thecommon beam 1007 to the optics instruments 417′ of the various flowcytometry units 1003. In the embodiment illustrated in FIGS. 106 and111, the guidance system 1273 includes a lower mirror assembly 1279mounted on one side wall 1071 of the housing 1009, an upper mirrorassembly 1281 mounted on the side wall 1071 above the lower mirrorassembly, and a series of reflecting filters 1283, one associated witheach optics instrument 417′. The lower mirror assembly is operable toreflect a beam 1007 from a suitable source upwardly to the upper mirrorassembly, and the upper mirror assembly is operable to reflect the beamthrough an opening in the side wall 1071 to the reflecting filters 431′of the various instruments 417′.

In one embodiment, the lower mirror assembly includes a base 1285fastened to the side wall 1071 of the housing 1009, a stage 1289 movablevertically on the base by a suitable mechanism 1291, such as amicrometer, a tiltable platform 1293 on the stage (e.g., a kinematicoptical mount Model P100-P available from Newport), and a mirror 1295 onthe platform, the position of the mirror being adjustable by moving thestage and the mirror platform to the appropriate locations. The uppermirror assembly is similar to the lower assembly, comprising a base1297, a vertically movable stage 1299, a tiltable platform on the stage1301, and a mirror 1303 on the platform. A pair of target plates 1309are affixed to the side wall of the housing 1009 between the upper andlower mirror assemblies. The target plates 1309 have vertically alignedholes 1311 therein to facilitate adjustment of the upper and lowermirrors so that an incoming beam 1007 is precisely reflected toward thereflecting filters 431′ of the instruments 417′, all of which filtersare aligned with the incoming beam.

Each of the first three reflecting filters 1315, 1317, 1319 functions asa beam splitter, i.e., it functions to reflect a specified percentage ofthe beam and to pass the remaining percentage of the beam. For example,in the case of four epi-illumination instruments, the reflecting filters431′ of the first three instruments each reflect a percentage of thelaser light 1007, so that each of the first three units of the seriesreceives 25% of the electromagnetic radiation of the original beam 1007.For example, the reflecting filters of the first, second and third unitsmay reflect 25%, 33% and 50% of the incident light, respectively. Thelast reflecting filter 1321 of the series desirably reflects all of theremaining light (about 25% of the original beam) to the last instrumentof the series. As a result, each of the four instruments should receivethe same intensity of radiation (light) to interrogate the cells inrespective streams.

Depending on the beam splitting devices used in the above system 1273,it may be desirable that the laser beam have a particular polarization.The transmittance-to-reflectance ratio of dielectric filters can varydepending on the polarization of the light. Further, when dealing withlinearly polarized light, dielectric filters (which are manufactured foruse at a specified angle of incidence) can be too sensitive tovariations in the angle of incidence. Circularly or ellipticallypolarized light alleviates this problem to some extent because thepolarization vector of the light is in a variety of differentorientations with respect to the optical axis of a dielectric filter asthe light interacts with the filter. Thus, elliptically or circularlypolarized light simulates randomly polarized light, which provides moretolerance for variations in the angle of incidence on a dielectricfilter. Accordingly, if the laser described above generates a beam oflight having a vertical polarization, for example, it may beadvantageous to convert the light to circularly polarized light beforeit is split. As will be understood by those skilled in the art, this canbe accomplished by passing the beam through a ¼-wave retardation plate(filter) of polarizing material having its optical axis rotated 45degrees relative to the plane of the laser polarization. The beam thustransmitted by the waveplate will have approximately circularpolarization, and it can be more easily split to provide multiple beamsto the optics systems of respective flow cytometer units.

Moreover, by rotating the wave retardation plate to alter the anglebetween the laser polarization and the optical axis of the material usedto make the waveplate, eccentricity can be introduced into theapproximately circular polarization of the beam (i.e., the polarizationcan be made more elliptical). Changing the eccentricity of theelliptical polarization of the beam can change thetransmittance-to-reflectance ratio of the dielectric filters by causingthe polarization vector for a greater percentage of the light to have aparticular angle with respect to the optical axis of the dielectricfilter. Accordingly, if the balance of light among the multiplecytometry units is outside the desired range, one can rotate thewaveplate to increase or decrease the eccentricity of the ellipticallypolarized light, thereby altering the transmittance-to-reflectanceratios of the various filters until a better balance is achieved.Similarly, if the waveplate is transmitting elliptically polarizedlight, one can influence the transmittance-to-reflectance ratio of oneof the filters by rotating that filter.

Regardless of the method used to split the single beam into multipleseparate beams. Balance of the power delivered to each cytometry unitcan be achieved by selectively blocking a percentage of the light tobring all the cytometry units down to the same level of power. Forexample, the neutral density filter 447′ of each epi-illumination system415′ can be selected to block more or less of the light to balance theilluminating power delivered by the beam splitting and guidance systemto each individual cytometry unit. If one channel of a multi-channelunit receives significantly more illumination from the beam splittingand guidance system, a neutral density filter 467′ that transmits lesslight can be used in the epi-illumination system 415′ of that channel tobring the illumination power for that channel more in line with theother channels. It is desirable, though not essential, thatchannel-to-channel variations in the illuminating power be less thanabout 10%. It is even more desirable that the channel-to-channelvariations be less than about 5%.

It will also be appreciated that pulsed laser scanning, as describedabove, may be desirable for multi-channel flow cytometry. For example,the UV multiline laser can be replaced with a mode-locked pulsed laseroperating at about 85 MHz to allow more flow cytometry channels to bepowered by a single laser. For example, the peak power provided in eachpulse of a mode-locked laser emitting pulses having a width (duration)of about 12 picoseconds at a frequency of about 85 MHz is approximately800 times the average power output of the laser. Thus, a mode-lockedlaser (e.g., a Vanguard 350 from Spectra-Physics) can provide enoughillumination energy to operate a few dozen cytometry units (e.g., 32cytometry units) while operating at only about 350 milliwatts.

The use of fiber optics for supplying light to the units is alsocontemplated. In this embodiment, fibers are used to direct light fromthe laser to respective units, thus eliminating the need for theguidance system described above.

F. Common Deflector Plates

In the embodiment shown in FIGS. 106 and 108-116, the sorting system119′ of each flow cytometry unit 1003 is substantially identical to thesorting system 119 described in the first embodiment, except that theunits desirably share two common deflector plates 1331 extending acrossthe width of the housing 1009 at the front of the housing. There areadvantages to using a common set of deflector plates, including aconsistent charge from one channel to the next, the use of a commonpower supply, a larger plate area providing a more stable electric fieldand more uniform droplet deflection, and a consistent angle ofdeflection for collection of sorted samples. The deflector plates 1331are mounted on a frame 1333 fastened to the housing 1009. Alternatively,separate plates could be provided for each unit.

G. Common Collection System

In the embodiment shown in FIGS. 107 and 116, a common collection system2801 includes two intercepting devices for each cytometry unit asdescribed above in connection with the collection system 2201 for thesingle unit. However, a common frame 2803 is provided to hold all eightof the intercepting devices. Also, one of the two intercepting devicesfor each cytometry unit directs fluid into a common waste trough 2805rather than a collection vessel. The waste trough makes it easier todiscard sorted droplets that contain particles of little value (e.g.,Y-chromosome bearing sperm cells for breeding dairy cows). If it isdesirable to retain all the sorted droplets, the waste trough can beremoved and collection vessels can be placed under each interceptingdevice. The four collection vessels in the embodiment shown in FIGS. 107and 116 rest in openings in the surface of a collection tray 2807. Acommon water bath (not shown) may be provided under the surface of thecollection tray to control the temperature of the contents of thecollection vessels.

H. Multi-Channel Control

The various flow cytometry units are controlled by the microprocessor131′, (or other suitable processing system) which desirably has a commoninput and a common output as discussed above.

Desirably, the operational parameters of each flow cytometry unit 1003can be set independently of the other units so that such parameters canbe varied as between units. These parameters may include, for example,the frequency of droplet formation, the control and sorting strategiesutilized by a particular unit, the criteria used by each unit toclassify and sort particles in the fluid supplied to the unit, and otherparameters. For example, in certain situations it may be desirable tosupply one or more units with carrier fluid 17′ at a first flow rate andother units a second (different) flow rate. Similarly, it may bedesirable to use one control sorting strategy (e.g., a “high efficiency”strategy) for one or more units while using a different strategy (e.g.,a “low loss” strategy) for other units. By controlled variation of theseparameters among the units, based on historical data and data collectedon a real-time basis, the throughput of the units can be managed and theresults of the system optimized. The capability of independent operationalso allows selected units to be operated in the event fewer than all ofthe units are needed or available.

I. Operation of Multi-Channel System

The operation of the multi-channel system of this embodiment is similarto that described previously, except that the multiple flow cytometryunits are adapted to conduct flow cytometry operations in parallel(i.e., during the same time period or overlapping time periods) forhigher throughput.

Prior to the start of a run, the fluid delivery system 1021 is flushed,if necessary, with cleaning solution from the tank 1173 by moving thevalve V5 to its cleaning position. The system is then conditioned withbuffer fluid using the syringe pump 1141. During this procedure, thevalves V1A-V1D and V2A-V2D are moved to establish communication with thewaste receptacle 1223 which is under vacuum. As a result, the cleaningsolution and/or buffer fluid flows through the system to waste. Thisprocess cleans the system 1021, primes the syringe pump 1141 and removesair bubbles from the system.

With the three-way valve 1149 suitably positioned, the syringe pump 1141is operated through an intake stroke to aspirate a quantity of carrierfluid 17′ containing particles, e.g., sperm cells, following which thevalve 1149 is moved to establish communication with the manifold 1177and the syringe pump moves through a discharge stroke to pump a volumeof carrier fluid into the sample reservoir 1207 to fill it. Thetemperature of the carrier fluid 17′ is controlled by the temperaturecontrol system 1257 to maintain the cells in the carrier fluid at thedesired temperature. With the valves V1A-V1D positioned to establishcommunication with the sample reservoir 1207, further operation of thesyringe pump 1141 forces carrier fluid through the lines to the needlesof respective nozzle assemblies for flow through the nozzles 101′, aspreviously described. At the same time, and with the valves V2A-V2Dpositioned to establish communication with the sheath fluid tank 1155,sheath fluid 19′ is forced through the supply lines to respective flowbodies and through the nozzles, also as previously described. Thisprocess continues for an appropriate length of time sufficient to pump asuitable volume of fluid through the system 1001. The duration of aparticular run will vary depending on the quantity of carrier fluid inthe supply vessel, the rate at which the carrier fluid is pumped throughthe system, and the number of channels in the system. For example, a runmay continue for only a limited period of time (e.g., 15 minutes duringwhich about one ml of carrier fluid is delivered to each nozzle) or itmay continue indefinitely, with the supply of fluid being replenished asneeded.

In the event a needle 157′ becomes clogged, the appropriate valve V1 ismoved to establish communication with the waste receptacle 1223. Sheathfluid 19′ entering the flow body 133′ will then flow under the force ofthe vacuum 1225 back through the needle 157′ to waste, thus flushing andclearing the needle. If there is a need to shut off the flow to aparticular nozzle, the valves V1 and V2 are simply switched to theirwaste positions.

Although the system described herein with respect to both the singlechannel and multi-channel configurations has been described with regardto particle separation, such as the separation of X and Y cells, it iscontemplated that such particles include any particles having differentcharacteristics which may be arbitrarily noted as characteristic A andcharacteristic B. Further, it will be understood that in someembodiments, the sorting function can be eliminated entirely, so thatthe flow cytometry apparatus (single-channel or multi-channel) operatesonly to classify the particles and not to sort them.

While the multi-channel system is described above in the context ofoperating the flow cytometry units in parallel, it will be understoodthat the units could also be operated in series. For example, it iscontemplated that particles in one stream could be sorted by one unitinto multiple populations, and that one or more of such sortedpopulations could then be passed through one or more other units inseries to perform additional sorting operations to sort differentparticles using the same or different sorting strategies.

J. Upright Multi-Channel Embodiment

FIGS. 119 and 120 show another exemplary multi-channel flow cytometrysystem. This system, generally designated 4001 comprises four cytometryunits 4009 ganged together. The nozzle system 101′, epi-illuminationoptics system 450′, deflector plates 629′, sample station 4051,contamination prevention mechanism 4031 and other components of eachunit 4009 are mounted on a shared vertical mounting board 4011.Referring to FIG. 120, a single laser 4013 and a beam splitting andguidance system 4015, which is substantially similar to the beamsplitting and guidance system 1273 described above, provide illuminationfor each epi-illumination system 450′. The laser 4013 passes through ahole 4019 (FIG. 119) in a common housing 4021 containing the beamsplitting and guidance system 4115. The beam splitting and guidancesystem 4115 and epi-illumination systems 450′ are on one side of theboard 4011. The focusing lens assembly 491′ of each epi-illuminationsystem 450′ extends through the board 4011 to the other side (similarlyto the configuration show in the single channel system shown FIGS. 26 &27), on which the remainder of the components for the units 4009 aremounted.

The units 4009 are all oriented so that their nozzle systems 101′ directthe fluid streams 21′ downward. Each unit 4009 also has a collectionsystem 4031, which includes a collection vessel 4033 for collectingdroplets 33 containing a desired population of particles and a wastecontainer 4035 for collecting other droplets 33. A water bath (notshown) or other temperature control may be used to control thetemperature of the collection vessel 4033.

The multiple flow cytometry units 4009 can also share a common powersupply (not shown), a common input for controlling operation of theunits (not shown), and a common output (not shown) allowing comparativeevaluation of the operation of the units 4009 relative to one another.As demonstrated by comparison of the two exemplary multi-channelembodiments 1001, 4001, the nature of the integrated platform and thesharing of features between or among multiple flow cytometry units in amulti-channel system can be varied extensively without departing fromthe scope of the present invention.

Impact of Multi-Channel Processing on Overall Process

The overall process described above can be performed with multi-channelsperm sorting to decrease the time required to sort the sperm cells.With few exceptions, the method does not change. One minor change isthat sorted sperm cells will be collected in multiple collectionvessels. The contents of the multiple collection vessels can be combinedfor concentration if desired. Alternatively, the contents of eachcollection vessel can be concentrated separately. It will be appreciatedthat the time required to sort a batch of sperm cells (e.g., anejaculate) from collection to completion of the cryopreservation stepcan be significantly reduced by using multiple cytometry units toprocess the batch. For example, if four cytometry units operate inparallel to process the batch of sperm cells, the time required tocomplete sorting is reduced to approximately one quarter of the timerequired to sort the batch using a single cytometry unit. Thus, bysubstituting the step of sorting sperm with four cytometry unitsoperating in parallel with the step of sorting sperm with a singlecytometry unit, the exemplary timeline for completion of the method fromcollection to completion of the freezing step can be reduced. The timecan be reduced even further by increasing the number of cytometersoperating in parallel to sort the sperm cells in the sample, subject tothe practical limitations involved in operating a parallel system havingmore than four such units. Thus, according to one embodiment of thepresent invention, the sorting step in the overall process describedabove is performed by sorting the sperm cells according to a specifiedDNA characteristic in a multi-channel flow cytometry apparatus. In yetanother embodiment, a sperm processing method comprises the step ofsorting sperm cells according to a specified DNA characteristic in amulti-channel flow cytometry apparatus in which each channel collects inthe range of about 2,000-10,000 sperm cells having a desired DNAcharacteristic per second.

Multi-Channel Sorting Example I

Bull semen was collected from a sexually mature bull using an artificialvagina and the sample transported to a nearby staining facility in atemperature-controlled container at 37° C. Upon receipt, the semen wasanalyzed for concentration, visual motility, motility and progressivemotility by the Hamilton-Thorn Motility Analyzer (IVOS), according tostandard and well known procedures (Farrell et al. Theriogenology, 49(4): 871-9 (Mar. 1998)).

Six tubes of 1 mL of 150×10⁶ sperm/mL sperm suspension were prepared bysuspending an aliquot of semen in 41° C. TCA #2 buffer containing 10 mMpyruvate bringing the overall pH to 7.35. Then varying amounts of 10 mMHoechst 33342 solution in water were added to the sperm samples to yieldfinal dye concentrations of 200, 300, 400, 500, 600, & 700 μM Hoechst33342. Each of the six samples was incubated at 41° C. for approximately30 minutes. The samples were analyzed by flow cytometry and the % CV ofthe X cell population was estimated by iterative computer algorithm forthe 200, 300, and 400 μM Hoechst 33342 samples. The % CV for the 300 and200 μM Hoechst 33342 were both ascertained to be within the acceptablerange near 1.3% CV. Accordingly, it was determined that a concentrationof 250 μM Hoechst 33342 would be used to stain a batch of sperm cellsfor further processing.

Two tubes containing 2 mL each of 150×10⁶ sperm/mL sperm suspension wereprepared by suspending an aliquot of semen in 41° C. TCA #2 buffercontaining 10 mM pyruvate (again bringing the overall pH to 7.35). Then10 mM Hoechst 33342 solution in water was added to each of the two spermsuspensions to yield a final dye concentration of 250 μM Hoechst 33342.The sperm suspensions were maintained in a 41° C. water bath for 30 min.After 30 minutes, the sperm suspensions were removed from the 41° C.water bath and 4 μL of 25 mg/mL FD&C #40 was added to one of thesuspensions. The other was stored at ambient temperature to providecomparison samples for the assessment assays.

The stained and quenched sperm suspension was loaded onto the sampleport of one channel of a four channel droplet sorting flow cytometer.Delbecco's PBS was used as the sheath fluid. The cytometer was equippedwith an orienting nozzle as described above and having a 60 micronorifice. A semicircular baffle plate was installed perpendicular to thelongitudinal axis of the nozzle as described above. The transducer wasoperated at 54 KHz and the droplet break-off location was controlledmanually. An epi-illumination optics system as described above was usedto direct approximately 25% of the beam of a continuous wave laser tointersect the fluid stream at a perpendicular angle. The focusing andcollection lens had a 0.65 numerical aperture. The beam was focused to aspot having a width less than 3 μm for slit scanning the sperm cells.Digital signal processing was used to extract the critical slopedifference and pulse area for each detected pulse waveform.Classification parameters for classification of X cells, Y cells, andundetermined cells in the two-dimensional CSD and pulse area featurespace were manually entered into the processing system for classifyingsperm cells according to chromosome content.

Sperm were sorted according to X and Y chromosome content using acoincidence accept sort strategy for collection of X cells, assigning a50/50 probability that each unclassified sperm was an X cell or Y cell.The sample fluid rate was manually adjusted to maintain purity ofcollected X cell population (as indicated by the GUI) at 85% or betterand to maintain the rate of X cell collection above a minimum rate.After approximately fifteen million X sperm had been collected in a tubethat had been soaked in sheath fluid for at least one hour and thencoated with 0.5 mL of 10% egg yolk in TCA #2 buffer at pH 7.0, the tubewas removed and replaced with an additional tube that has been similarlyprepared.

Immediately after removing a collection tube from the flow cytometer, acomparison sample from the stained, but not sorted, sperm suspension wasprepared. The sorted and comparison samples were centrifuged for 7 min @750 g in a 15 mL tube. The supernatants were removed using a transferpipette to yield a concentration of approximately 40 million sperm/mL.TCA #2 buffer pH 7.0 was added to the sperm suspensions to yield a finalconcentration of approximately 20 million sperm/mL. This processcontinued until the flow cytometer had produced four collection tubes(A2-A5). The sorted samples and “non-sorted” comparison samples wereassessed by IVOS. Sorted sample A3 and its non-sorted comparison samplewere tested for % intact acrosomes by differential interference contrastmicroscopy. All the sorted samples were counted by hemacytometer todetermine the output rate of sorted sperm per hour. The % X chromosomebearing sperm was confirmed by flow cytometer reanalysis. Results of theIVOS assessment for the sorted and “non-sorted” comparison samples areprovided in FIGS. 121 (motility) and 122 (progressive motility). Thetotal number of sperm sorted into each collection tube is shown in FIG.123. The rate of sperm sorted per hour for each collection period isshown in FIG. 124. Percentage of X chromosome bearing sperm for eachsorted sample is listed in FIG. 125. Results of the assessment of theacrosome integrity were 72% intact acrosomes for the sorted sample and78% for the non-sorted comparison sample.

The results demonstrate the technical ability to yield more than 5,000sorted X cells per second at greater than 85% purity per channel ofmulti-channel flow cytometry system for sustained periods. The resultsalso show the technical ability to yield more than 7,000 X cells persecond at greater than 85% purity for sustained periods under idealconditions. Further, the results indicate that samples of sorted spermcells obtained by such high-speed flow cytometric sorting will sufferonly slight declines in motility, indicating that the sorted sperm willhave good fertility.

Multi-Channel Sorting Example II

Bull semen was collected from a sexually mature bull using an artificialvagina. The ejaculate was split into two aliquot. The first aliquot of250 μL of semen was suspended in 5 mL of 37° C. Triladyl®. The secondaliquot, which comprised the remained of the ejaculate, was suspended intwo parts 37° C. carbonate buffer (pH 6.1-6.2). Both aliquots weretransported at 37° C. in a temperature-controlled container to aprocessing facility. At the processing facility, the first aliquot wasfloated in ˜120 mL of 37° C. water in a 200 mL beaker and placed in acold room to slowly cool to 5° C. The second aliquot was analyzed forconcentration, motility and progressive motility by the Hamilton-ThornMotility Analyzer (IVOS), according to standard and well knownprocedures (Farrell et al. Theriogenology, 49 (4): 871-9 (Mar. 1998)).

Three 1 mL tubes of 150×10⁶ sperm/mL sperm suspension were prepared bytransferring sub-aliquots containing 150 million sperm from the secondaliquot to empty tubes, centrifuging at 500 g for 5 min, removing thesupernatants, and re-suspending the sperm pellets in 1 mL of 28° C. TCA#2 buffer containing 10 mM pyruvate pH 7.35. Ten mM Hoechst 33342solution in water was added to each of the three tubes in variousamounts to yield final dye concentrations of 100, 150, & 200 μM Hoechst33342. Each of the three tubes was held at 28° C. for approximately 60minutes. Sperm from each of the three tubes was analyzed by flowcytometry and the CV of total fluorescence intensity of the X populationwas determined for the 100, 150, and 200 μM Hoechst 33342 stainingconditions using an interactive computer algorithm. The CVs for the 150and 200 μM Hoechst 33342 were both within the acceptable range near1.3%. Thus, it was determined to use staining conditions including 150μM Hoechst 33342 concentration for sorting.

One tube containing 5 mL of 150×10⁶ sperm/mL sperm suspension wasprepared by transferring a sub-aliquot containing 750 million sperm fromthe second aliquot, centrifuging at 500 g for 5 min, removing thesupernatant, and re-suspending the sperm pellet in 28° C. TCA #2 buffercontaining 10 mM pyruvate (pH 7.35). Ten mM Hoechst 33342 solution inwater was added to the tube in an amount yielding a final dyeconcentration of 150 μM Hoechst 33342. The tube was maintained in a 28°C. water bath for 60 min. After 60 minutes, the tube was removed fromthe 28° C. water bath and 10 μL of 25 mg/mL FD&C #40 was added.

The now stained and quenched sperm suspension was loaded onto the sampleport of one channel of a multi-channel droplet sorting flow cytometersystem. The sperm suspension was maintained at 28° C. Usingsubstantially the same instrument settings as set forth in Multi-channelExample I, X & Y chromosome bearing sperm were separated by the flowcytometry system using a coincidence abort sort strategy for a periodnecessary to place an enriched X cell population of approximatelyeighteen million sperm into a collection tube that had been prepared bysoaking with sheath buffer for at least one hour and then adding 0.5 mLof Triladyl® cryo-preservation media containing 10 mM pyruvate pH 6.6.The sperm cells were introduced into the flow cytometry system at a rateof between about 25,000 and 30,000 cells/second. An enriched populationof X cells was collected at a rate varying from 4,500 per second to6,000 per second. When approximately eighteen million sperm had beensorted into a collection tube, the tube was removed and replaced withanother tube that had been similarly prepared. Immediately after removalof a collection tube from the flow cytometer, the sorted spermsuspension was centrifuged for 7 min @ 700 g. The supernatant wasremoved using a transfer pipette to yield a concentration ofapproximately 100 million sperm/mL. Triladyl® cryo-preservation mediacontaining 10 mM pyruvate (pH 6.6) was added to the sperm suspensions toyield a final concentration of approximately 50 million sperm/mL. Thisprocess continued until the flow cytometer had produced three collectiontubes (D1-D3). Approximately 52 million sperm were sorted in 259 minyielding an overall collection rate of about 12 million enriched X spermper hour of sorting. The re-suspended sorted sample tubes were floatedin ˜120 mL of 28° C. water in a 200 mL beaker and placed in a 5° C. coldroom to slowly cool.

After the sorted samples reached 5° C., the three tubes of sorted spermwere combined into one tube. The pooled sample was analyzed by IVOS todetermine the % motility, % progressive motility, and concentration.Additional Triladyl® cryo-preservation media containing 10 mM pyruvatepH 6.6 was added to the sample to yield a final concentration ofapproximately 50 million sperm per mL. The % X-chromosome bearing spermin the sorted pooled sample was 87% as determined by flow cytometerre-analysis. A summary of the IVOS assessment compared to the non-sortedsample of the same ejaculate is illustrated in FIG. 126.

The pooled sorted sample and the first aliquot were loaded into standard0.25 cc straws in a 5° C. cold room. The loaded straws were transferredto a programmable freezer and frozen by the following program: 5 min @5° C., cool from 5° C. to −12° C. @ 4° C./min, cool from −12° C. to−100° C. @ 40° C./min, cool from −100° C. to −140° C. @ 20° C./min, holdat −140° C. After the straws had reached −140° C., they were quicklyremoved from the freezer and plunged into liquid nitrogen.

Thawed straws were analyzed by IVOS for % motility and % progressivemotility after incubation at 37° C. for 30 and 120 minutes. Results froma set of two sorted and unsorted straws are summarized in FIG. 127 andFIG. 128.

Multi-Channel Sorting Example III

Bull semen was collected from a sexually mature bull using an artificialvagina and the ejaculate split into two aliquot. A first aliquot of 250μL of semen was suspended in 5 mL of 37° C. Triladyl®. A second aliquot,which comprised the remainder of the ejaculate, was suspended in twoparts 37° C. carbonate buffer (two parts 0.097 moles/L of NaHCO₃, 0.173moles/L of KHCO₃, 0.090 moles/L C₆H₈0₇H₂O in water) (pH 6.1-6.2). Bothaliquots were transported at 37° C. in a temperature-controlledcontainer to the processing facility. At the processing facility, thefirst aliquot was floated in ˜120 mL of 37° C. water in a 200 mL beakerand placed in a cold room to slowly cool to 5° C. The second aliquot wasanalyzed for concentration, motility and progressive motility by theHamilton-Thorn Motility Analyzer (IVOS), according to standard and wellknown procedures (Farrell et al. Theriogenology, 49 (4): 871-9 (Mar.1998)).

Two tubes of 150×10⁶ sperm/mL sperm suspension were prepared bytransferring into each of two empty tubes a fraction containing 900million sperm from the second aliquot, centrifuging each tube at 500×gfor 5 minutes, removing the supernatant from each tube, andre-suspending each sperm pellet in 6 mL of 28° C. TCA #2 buffercontaining 10 mM pyruvate (pH 7.35). 10 mM Hoechst 33342 solution inwater was added to each of the two tubes to yield final dyeconcentrations of 200 μM Hoechst 33342 in one tube and 400 μM Hoechst33342 in the other tube. Each of the two tubes was held at 28° C. forapproximately 120 minutes. Sperm from each of the tubes was analyzed byflow cytometry and the CV of total fluorescence intensity of the Xpopulation was determined for the 200 μM and 400 μM Hoechst 33342staining conditions using an interactive computer algorithm. The CVs forthe 200 μM and 400 μM Hoechst 33342 were both within the acceptablerange of about 1.3%. The sperm suspension stained with a concentrationof 200 ptM Hoechst 33342 was chosen for sorting. 10 μL of 25 mg/mL FD&C#40 was added to this tube of stained sperm suspension just prior tosorting.

The stained sperm suspension was loaded onto the sample port of onechannel of a multi-channel droplet sorting flow cytometer system. Thesperm suspension was maintained at 28° C. Using substantially the sameinstrument settings as set forth in Multi-channel Example I, X & Ychromosome bearing sperm were separated by the flow cytometry systemusing a coincidence abort sort strategy for a period of time necessaryto place an enriched X chromosome bearing cell population ofapproximately eighteen million sperm into a collection tube that hadbeen prepared by soaking with sheath buffer for at least one hour andthen adding 0.5 mL of Triladyl® cryo-preservation media (pH 6.6). Thesperm cells were introduced into the flow cytometry system at a rate ofbetween about 25,000 and 30,000 cells/second. An enriched population ofX chromosome bearing cells was collected at a rate varying from 4,500per second to 6,000 per second. When approximately eighteen millionsperm had been sorted into a collection tube, the tube was removed andreplaced with another tube that had been similarly prepared. Immediatelyafter removal of a collection tube from the flow cytometer, the sortedsperm suspension was centrifuged for 7 min @ 700×g. The supernatant wasremoved using a transfer pipette to yield a concentration ofapproximately 100 million sperm/m L. Triladyl® cryo-preservation media(pH 6.6) was added to the sperm suspensions to yield a finalconcentration of approximately 50 million sperm/mL. This processcontinued until the flow cytometer had produced two collection tubes(C1-C3). Approximately 35 million sperm were sorted in 193 minutesyielding an overall collection rate of 11 million enriched X chromosomebearing cells per hour of sorting. The re-suspended sorted sample tubeswere floated in ˜120 mL of 28° C. water in a 200 mL beaker and placed ina 5° C. cold room to slowly cool.

After the sorted samples reached 5° C., the three tubes of sorted spermwere combined into one tube. The pooled sample was analyzed by IVOS todetermine the % motility, % progressive motility and concentration.Additional Triladyl® cryo-preservation media (pH 6.6) was added to thesample to yield a final concentration of approximately 50 million spermper mL. The % X-chromosome bearing sperm in the sorted pooled sample was88% as determined by flow cytometer re-analysis. A summary of the IVOSassessment compared to the non-sorted sample of the same ejaculate isillustrated in FIG. 129.

The pooled sorted sample and unsorted sample (i.e., the first aliquotfrom above) were loaded into standard 0.25 cc straws in the 5° C. coldroom. The loaded straws were transferred to a programmable freezer andfrozen by the following program: 5 min @ 5° C., cool from 5° C. to −12°C. @ 4° C./min, cool from −12° C. to −100° C. @ 40° C./min, cool from−100° C. to −140° C. @ 20° C./min, hold at −140° C. After the straws hadreached −140° C., they were quickly removed from the freezer and plungedinto liquid nitrogen.

Thawed straws were analyzed by IVOS for % motility and % progressivemotility after incubation at 37° C. for 30 and 120 minutes. Results froma set of sorted and unsorted straws are summarized in FIG. 130 and FIG.131.

Multi-Channel Sorting Example IV

Bull semen was collected from a sexually mature bull using an artificialvagina and the ejaculate split into two aliquot. The first aliquot of250 μL of semen was suspended in 5 mL of 37° C. Triladyl®. The secondaliquot, which comprised the remained of the ejaculate, was suspended intwo parts 37° C. carbonate buffer (two parts 0.097 moles/L of NaHCO₃,0.173 moles/L of KHCO₃, 0.090 moles/L C₆H₈O₇.H₂O in water) (pH 6.1-6.2)and held under CO₂. Both aliquots were transported at 37° C. in atemperature-controlled container to the processing facility. At theprocessing facility, the first aliquot was floated in ˜120 mL of 37° C.water in a 200 mL beaker and placed in the cold room to slowly cool to5° C. The second aliquot was analyzed for concentration, motility andprogressive motility by the Hamilton-Thorn Motility Analyzer (IVOS),according to standard and well known procedures (Farrell et al.Theriogenology, 49 (4): 871-9 (Mar. 1998)).

A 5 mL tube of 150×10⁶ sperm/mL sperm suspension was prepared bytransferring a fraction containing 750 million sperm from the secondaliquot (pH 6.1-6.2) to an empty tube and adding 28° C. carbonate buffer(pH 7.35) to a final volume of 5 ml. To this sperm suspension, 10 mMHoechst 33342 solution in water was added to yield a final dyeconcentration 150 μM Hoechst 33342. The suspension was held at 41° C.under CO₂ for approximately 40 minutes and then placed at 28° C. forsorting. Ten μL of 25 mg/mL FD&C #40 was added to the tube of stainedsperm suspension just prior to sorting.

The stained sperm suspension was loaded onto the sample port of onechannel of a multi-channel droplet sorting flow cytometer system. Thesperm suspension was maintained at 28° C. X & Y chromosome bearing spermwere separated by the flow cytometry using a coincidence abort sortstrategy for a time period necessary to place an enriched X chromosomebearing cell population of approximately eighteen million sperm into acollection tube that had been prepared by soaking with sheath buffer forat least one hour and then adding 0.5 mL of Triladyl® cryo-preservationmedia (pH 6.6). The sperm cells were introduced into the flow cytometrysystem at a rate of between about 25,000 and 30,000 cells/second. Anenriched population of X chromosome bearing cells was collected at arate varying from 4,500 per second to 6,000 per second. Whenapproximately eighteen million sperm had been sorted into a collectiontube, the tube was removed and replaced with another tube that has beensimilarly prepared. Immediately after removal of a collection tube fromthe flow cytometer, the sorted sperm suspension was centrifuged for 7min @ 700×g. The supernatant was removed using a transfer pipette toyield a concentration of approximately 100 million sperm/mL. Triladyl®cryo-preservation media and pyruvate (pH 6.6) was added to the spermsuspensions to yield a final concentration of approximately 50 millionsperm/mL. This process continued until the flow cytometer had producedtwo collection tubes (C2-C3). The re-suspended sorted sample tubes werefloated in ˜120 mL of 28° C. water in a 200 mL beaker and placed in a 5°C. cold room to slowly cool.

After the sorted samples reached 5° C., the two tubes of sorted spermwere combined into one tube. The pooled sample was analyzed by IVOS todetermine the % motility, % progressive motility and concentration.Additional Triladyl® cryo-preservation media and pyruvate (pH 6.6) wasadded to the sample to yield a final concentration of approximately 50million sperm per mL. A summary of the IVOS assessment compared to thenon-sorted sample of the same ejaculate is illustrated in FIG. 132.

The pooled sorted sample and unsorted sample (i.e., the first aliquotfrom above) were loaded into standard 0.25 cc straws in the 5° C. coldroom. The loaded straws were transferred to a programmable freezer andfrozen by the following program: 5 min @ 5° C., cool from 5° C. to −12°C. @ 4° C./min, cool from −12° C. to −100° C. @ 40° C./min, cool from−100° C. to −140° C. @ 20° C./min, hold at −140° C. After the straws hadreached −140° C., they were quickly removed from the freezer and plungedinto liquid nitrogen.

Thawed straws were analyzed by IVOS for % motility and % progressivemotility immediately after thawing and after incubation at 37° C. for 30minutes. Results from a set of sorted and unsorted straws are summarizedin FIG. 133 and FIG. 134.

Capillary Tube Nozzle System

FIG. 135 illustrates an alternative nozzle system, generally designated1335, similar to that described above except that a capillary tube 1337(of quartz or fused silica, for example) is connected to the nozzle 137so that fluid exiting the nozzle orifice 103 is directed into andthrough the tube. The optics system 109 of the flow cytometer isoptically coupled to the side of the tube in a suitable manner, as by achamber 1341 filled with a light-transmitting medium such as oil or gelhaving a known index of refraction. The use of a capillary tube,compared to the jetting of the fluid stream through open space, has thebenefit of reducing the lensing of the stream 21 due to the acousticalenergy supplied by the transducer 105, and enabling the focusing lens1343 to be positioned immediately adjacent the fluid stream forincreasing resolution of the emission signals.

After the particles have been interrogated and classified, they may besorted using any conventional techniques known to those skilled in theart, as by use of a fluid switching device shown in FIG. 137 or othersuitable devices such as photo-damage systems or droplet sortingsystems.

Sorting Techniques Other than Droplet Sorting

Photo-Damage Sorting

The flow cytometry improvements of this invention are applicable notonly to droplet cell sorting as described above, but also to othersorting techniques, such as sorting by photo-damage (laser ablation).Photodamage sorting is discussed in U.S. Pat. No. 4,395,397, which isincorporated herein by reference in its entirety. FIG. 136 schematicallyillustrates one embodiment of a single-channel flow cytometryphoto-damage system, generally designated by the reference number.

As shown in FIG. 136, the photo-damage sorting system 1351 is similar tothe droplet sorting system of FIG. 2, and corresponding parts aredesignated by corresponding reference numbers with the addition of adouble prime (″). In general, the system comprises the same componentsas the system of FIG. 2, except that the droplet sorting components areeliminated (e.g., the transducer 105, the charging device 627, thedeflector plates 629, and associated power sources 635). Instead thesecomponents are replaced by a laser 1353 or similar device which isresponsive to instructions received from the microprocessor 131″ toablate undesired particles in the fluid stream 21″. As a result, thestream collected in a collection receptacle 1355 contains a desiredpopulation of particles. For example, if the particles being analyzedare sperm cells and the intended result is to collect sperm cells havinga characteristic A (e.g., a desired chromosome content), then themicroprocessor receives signals from the epi-illumination system 415″which identifies cells not having characteristic A and selectivelyactivates the laser to ablate such cells or otherwise render themineffective.

Different control sorting strategies can be employed in a photo-damagesystem, including the “high recovery” and “high purity” sortingstrategies discussed above in the context of a droplet sorter. In aphoto-damage system, particles contained in the fluid stream are spacedat various intervals along the stream and generally follow one afteranother in single file. The particles have different characteristics,some having a characteristic A, for example, and others having acharacteristic B. The sequence of particles is random, so viewed as acontinuous procession, the particles can be divided into differentparticle series, one following another, including a first particleseries consisting only of one or more particles having characteristic A,a second particle series consisting only of one or more particles havingcharacteristic B and a third particle series consisting of two or moreclosely spaced particles at least one of which has characteristic A andat least one of which has characteristic B. The latter (third) groupgenerally corresponds to the closely spaced particles in a “coincident”droplet discussed previously, at least for sorting strategy purposes.Thus, the two or more particles in the third group may be closely spacedin the sense that the spatial separation between the particles isinsufficient to allow accurate discrimination/classification of theparticles, or because such separation is insufficient to permit oneparticle in the series to be ablated by the laser without damaging theother particle(s) in the same series. In any event, the closely spacedparticles in each (or at least some) of the third series of particlescan be ablated or not abated, depending on the sorting strategyemployed. It should be noted that multiple particles in a first seriesor multiple particles in a second series could be “closely spaced”, butsince the particles in any such series have the same characteristic (Aor B), they are treated as a single-particle series, at least forsorting strategy purposes.

The photo-damage system can be a single-channel system or amulti-channel system, as described above.

Fluid Switching Sorting

It is contemplated that the principles of this invention can also beapplied to flow cytometry systems using fluid switching techniques, asdisclosed, for example, in U.S. Pat. No. 6,432,246 (Adair), U.S. Pat.No. 4,756,427 (Gohde, et al.), and U.S. Pat. No. 3,791,517 (Friedman),which are incorporated herein by reference in their entireties. FIG. 137is a partial view showing such a system, generally designated 1357. Itis substantially identical to the system shown in FIG. 2 except that thenozzle system 101″ includes a capillary tube 1369 (e.g., see FIG. 135),and the sorting system comprises a fluid-switching device 1359 coupledto the capillary tube 1369 downstream from the interrogation location115′. The construction and operation of the fluid-switching device canincorporate any conventional fluid switching technology such asdisclosed in the above-referenced patents. In general, the devicefunctions to sort desired particles from undesired particles in responseto instructions received from the processor by intermittently divertingportions of the fluid stream containing the desired/undesired particlesalong separate flow paths 1361, 1365 for collection in vessels or thelike. The switching is commonly achieved by selectively actuating atransducer 1367 in one of the flow paths.

The various sorting strategies described above in regard to dropletsorting and photo-damage sorting can also be employed in afluid-switching system. In the fluid-switching system, particlescontained in the fluid stream are also spaced at various intervals alongthe stream and generally follow one after another in single file. Theparticles have different characteristics, some having a characteristicA, for example, and others having a characteristic B, and the sequenceof particles is random. Therefore, as discussed above in regard to thephoto-damage system, the procession of particles can be divided intodifferent particle series, one following another, including a firstparticle series comprising one or more particles having characteristicA, a second particle series comprising one or more particles havingcharacteristic B and a third particle series comprising two or moreclosely spaced particles at least one of which has characteristic A andat least one of which has characteristic B. The latter (third) groupgenerally corresponds to the closely spaced particles in a “coincident”droplet discussed previously, at least for sorting strategy purposes.Thus, the two or more particles in the third group may be closely spacedin the sense that the spatial separation between the particles isinsufficient to allow accurate discrimination/classification of theparticles, or because such separation is insufficient to permit oneparticle in the series to be diverted by the fluid-switching deviceseparate from the another particle in the same series. In any event, theclosely spaced particles in each (or at least some) of the third seriesof particles can be diverted to one collection location or another,depending on the sorting strategy employed. As explained above inconnection with photo-damage sorting, multiple particles in a firstseries or multiple particles in a second series could be “closelyspaced”, but since the particles in any such series have the samecharacteristic (A or B), they are treated as a single-particle seriesfor the purpose of sorting strategy.

The fluid switching system can be a single-channel system or amulti-channel system, as described above.

Droplet Interference Sorting

It is also contemplated that the technology of this invention can beused in conjunction with a droplet interference fluidic switchingtechnique. For example, a high-speed droplet interference sorting system1371, shown schematically in FIG. 138, may be used to sort particles bydiverting selected segments of the coaxial carrier and sheath fluidstream.

In contrast to some other sorting techniques, the droplet interferencesorting technique does not require the coaxial carrier and sheath streamto be formed into droplets. Thus, there is no need to couple the nozzlesystem 101′″ used for delivery of the carrier and sheath fluids with adroplet generation system. By way of example only, passing the carrierand sheath fluids through a nozzle system at 60 psi to create a 50micron diameter stream is one suitable arrangement for formation of alaminar coaxial fluid stream for delivery of particles to the dropletinterference sorting system. Particles in the coaxial fluid stream areanalyzed and classified by the optics system 109′″ and processor 131′″as they move through the interrogation location 115′″, as has beendescribed above for the other sorting systems. Sorting occurs downstreamfrom the interrogation location, at a location where the coaxial fluidstream intersects a high-speed droplet interference stream 1373.

The droplet interference stream 1373 is generated by a dropletgeneration system 1375 similar to the droplet generation system used fordroplet sorting. A high-speed fluid stream 1379 passes through ahigh-speed nozzle system 1377 that is coupled to a piezoelectrictransducer 1381 or other source of acoustical energy for causing thehigh-speed fluid stream to break into droplets 1383 downstream from thehigh-speed nozzle. For example, a particle-free fluid at 1500 psi may bepassed through the high-speed nozzle to form a 70 micron diameterhigh-speed fluid jet. The high-speed nozzle may be oscillated at 400 KHzto form high-speed droplets. The high-speed droplets 1383 pass throughan electric field generated by one or more electric deflection plates1387 so that the path of the high-speed droplets may be controlled byselectively applying an electric charge to the droplets, as was done tocontrol the path of droplets in the droplet sorting system. Thehigh-speed droplet interference stream is directed so some high-speeddroplets intersect the coaxial fluid stream at a point 1399 downstreamfrom the interrogation location. For example, uncharged droplets 1389may be directed to collide with the fluid stream while charged droplets1391 are deflected away from the coaxial fluid stream. When a high-speeddroplet collides with the coaxial fluid stream, a segment 1397 of thefluid stream and any particles contained therein are diverted from thepath they would have otherwise taken. The application of a charge or nocharge to a high-speed droplet may be timed so the arrival of thatdroplet at the intersection 1399 with the coaxial fluid stream coincideswith the arrival of a particular segment of the coaxial fluid stream.Thus, by selectively charging high-speed droplets depending on theclassification of particles contained within the coaxial streamsegments, one can sort particles by diverting all coaxial fluid streamsegments that contain one or more selected particles and not divertingother coaxial stream segments or vice-versa. Collection capillaries 1403having a slight vacuum may be used to collect both the diverted 1397 andundiverted coaxial stream segments. The droplet interference sortingsystem may be set up so the high-speed droplets merge with divertedcoaxial stream segments or so the high-speed droplets remain separatefrom the diverted stream segments after collision with the coaxialstream segments.

Because there are no particles or cells in the high-speed dropletinterference stream 1373, it is possible to use very high pressures andvery high droplet frequencies without damaging the particles or cells tobe sorted. This allows sorting of stream segments each having lessvolume (e.g., four times less volume) than the volume of a droplet inthe droplet sorting system. This greatly increases the maximumthroughput of the system while also reducing the dilution factor of thesorted particles. Moreover, because finely filtered liquid with no cellsor particles is used to form the droplet interference stream, moreconsistent droplet formation is possible because the droplet formationnozzle is less likely to become clogged or suffer from protein buildupthan the nozzle system used in the droplet sorting system. Anotheradvantage is that the distance between particle analysis at theinterrogation location and the sorting point 1399 can be reduced (e.g.,by a factor of four), allowing more accurate prediction of the time ofarrival of a particular particle at the sorting point. Furthermore, thedroplet interference system allows more flexibility in adjustment ofnozzle size or pressure for the coaxial fluid stream. If desired, thedroplet interference sorting system can be combined with the capillarytube nozzle system. A multi-channel droplet interference sorting systemmay use a high-pressure fluidic pump to supply multiple dropletinterference stream generating nozzles with fluid from a common fluidsupply.

When introducing elements of the present invention or the embodiment(s)thereof, the articles “a,” “an,” “the,” and “said” are intended to meanthat there are one or more of the elements. The terms “comprising,”“including,” and “having” are intended to be inclusive and mean thatthere may be additional elements other than the listed elements. Theterm “or” is intended to include “and/or” and is intended to mean “oneor another or both.” Thus, an indication of “ABC or DEF” means (1) ABC,or (2) DEF, or (3) both ABC and DEF. The term “and/or” is intended tohave the same meaning as “or” as defined above. Thus, the term “and/or”is intended to include “or” and is intended to mean “one or another orboth.” For example, an indication of “ABC and/or DEF” means (1) ABC, or(2) DEF, or (3) both ABC and DEF.

In view of the above, it will be seen that the several objects of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above constructions, products,and methods without departing from the scope of the invention, it isintended that all matter contained in the above description and shown inthe accompanying drawings shall be interpreted as illustrative and notin a limiting sense.

We claim:
 1. A multi-channel system for classifying sperm cellsaccording to one or more characteristics of the sperm cells, said systemcomprising: a plurality of flow cytometry units each of which isoperable to provide sex sorted sperm from a mixture of sperm cells byinterrogating a stream of fluid containing said sperm cells using a beamof electromagnetic radiation and detecting the response with at leastone sensor operable to generate a output signal indicative of at leastone characteristic of the sperm cells; said units sharing an integratedplatform comprising a common processor for detecting waveform pulsesfrom the at least one output signal from the at least one sensor,extracting features in the detected waveform pulses, and discriminatingthe detected waveform pulses as a function of their extracted feature.2. The system of claim 1 further comprising a common source ofelectromagnetic radiation comprising a plurality of laser pulses, eachpulse having a peak power that is greater than the average power outputof a laser and a wavelength in a range of about 350-370 nm.
 3. Thesystem of claim 2 wherein said laser emits pulses having a width ofabout 1-100 picoseconds at a pulse frequency of about 50-150 MHz at apower of about 50-500 milliwatts.
 4. The system of claim 2 wherein saidcommon source of said pulsed electromagnetic radiation comprises asingle pulsed laser beam.
 5. The system of claim 4 further comprising abeam splitting system for splitting the single pulsed laser beam intomultiple beams and directing the multiple beams into optics systems ofrespective flow cytometry units.
 6. The system of claim 1 wherein saidintegrated platform further comprises a common housing, said flowcytometry units comprising interchangeable modules removably mounted inthe housing.
 7. The system of claim 1 wherein said processor is operableto output an indication of the fluorescence intensity measured by eachunit.
 8. The system of claim 1 wherein said processor is operable tooutput an indication of a decision boundary used by each unit fordiscriminating between sperm cells.
 9. The system of claim 1 whereinsaid flow cytometry units operate in parallel.
 10. The system of claim 1wherein said integrated platform further comprises a common input forcontrolling operation of the flow cytometry units.
 11. The system ofclaim 1 wherein said plurality of flow cytometry units comprises ajet-in-air droplet sorting flow cytometry unit.
 12. The system of claim1 wherein the common processor receives and processes said informationto permit evaluation of the operation of one unit relative to anotherunit.
 13. The system of claim 1 wherein the processor is operable toprocess the information in real time.